JP2022516438A - Treatment guides and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof - Google Patents

Abstract

The subject of the present invention is angiotensin receptor agonists and / or precursors thereof for use in the treatment of disease in a subject, and the choice of disease is heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI). , Stroke, liver failure, burns, trauma, severe infections (microbial disease, viral disease (eg AIDS), parasitic disease (eg malaria)), SIRS or septicemia, cancer, acute renal disorder (AKI), CNS disorder ( It consists of groups including (eg, seizures, neurodegenerative diseases), autoimmune diseases, vascular diseases, hypotension, shock, and the subject has a predetermined range of DPP3 protein levels and / or DPP3 activity in body fluid samples. Over.

Description

The subject of the present invention is angiotensin receptor agonists and / or precursors thereof for use in the treatment of diseases in a subject.
-The diseases include heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial disease, viral disease (eg AIDS), parasite disease (eg AIDS)). Selected from groups including malaria))), SIRS or septicemia, cancer, acute renal failure (AKI), CNS disorders (eg, seizures, neurodegenerative disorders), autoimmune disorders, vascular disorders, hypotension, and shock.
The subject has an amount and / or DPP3 activity of DPP3 protein in a body fluid sample that exceeds a predetermined threshold.

Dipeptidyl peptidase 3 (also known as dipeptidyl aminopeptidase III, dipeptidyl aryl amidase III, dipeptidyl peptidase III, enkephalinase B, or erythrocyte angiotensinase, abbreviated as DPP3, DPPIII) A metallopeptidase that removes dipeptides from physiologically active peptides (such as enkephalin and angiotensin).

DPP3 was first identified in 1967 by Ellis and Nuenke from purified extracts of the anterior pituitary gland and its activity was measured. This enzyme is listed as EC 3.4.14.4, has a molecular weight of approximately 83 kDa and is highly conserved in eukaryotes and prokaryotes (Prajapati and Chauhan 2011). The amino acid sequence of the human variant is shown in SEQ ID NO: 1. Dipeptidyl peptidase III is a ubiquitously expressed predominantly cytosolic peptidase. Although the signal sequence is missing, several studies have reported membrane activity (Lee and Snyder 1982).

DPP3 is a zinc-dependent exopeptidase belonging to the peptidase family M49. DPP3 has broad substrate specificity for oligopeptides of various compositions consisting of 3/4 to 10 amino acids and can also cleave after proline. DPP3 is known to hydrolyze dipeptides from the N-terminus of its substrates (angiotensin II, III, and IV; including Leucine-enkephalin and Met-enkephalin; endomorphines 1 and 2). Metallopeptidase DPP3 has optimal activity at pH 8.0-9.0 and can be activated by the addition of divalent metal ions (such as Co ²⁺ and Mg ²⁺ ).

From the structural analysis of DPP3, the catalytic motifs HELLGH (hDPP3 450-455) and EECRAE (hDPP3 507-512), as well as amino acids important for substrate binding and hydrolysis: Glu316, Tyr318, Asp366, Asn391, Asn394, His568, Arg572 , Arg577, Lys666, and Arg669 (Prajapati and Chauhan 2011; Kumar et al. 2016; numbers are for the sequence of human DPP3; see SEQ ID NO: 1). Considering all known amino acid or sequence regions involved in substrate binding and hydrolysis, the active site of human DPP3 can be limited to the region between amino acids 316 and 669.

The most important substrate for DPP3 is angiotensin II (Ang II), the major effector of the renin-angiotensin system (RAS). RAS is a cardiovascular disease (Dostal et al., 1997 J Mol Cell Cardiol; Vol. 29: 2893-902; Roks et al., 1997 Heart Vessels. Suppl Vol. 12, pp. 119-24), and sepsis and septic shock. It is activated in (Correa et al., 2015 Crit Care 2015; Volume 19: Page 98). Ang II, in particular, has been shown to alter many cardiovascular functions, including blood pressure control and cardiac remodeling.

Although the exact biological function of DPP III in cell physiology is unknown, recent findings suggest a role not only in protein metabolism, but also in pain changes and inflammatory processes (Prajapati and Chauhan 2011). ). In addition, DPP3 reduced blood pressure in hypertensive mice infused with Ang II without altering heart rate (Pang et al., 2016). However, mice with normal blood pressure did not change their blood pressure when injected with DPP3.

The decrease in blood pressure by administering angiotensin receptor antagonist in combination with DPP3 to mice infused with Ang II was similar to the decrease in blood pressure by injection of DPP3 alone or by angiotensin receptor antagonist alone (Pang et al., 2016). Hypertension Vol. 68: pp. 630-41).

Angiotensin II (Ang II) is a peptide hormone naturally produced by the body that regulates blood pressure through vasoconstriction and sodium reabsorption. The hemodynamic effects of Ang II administration have been the subject of many clinical studies and have demonstrated significant effects on systemic and renal blood flow (Harrison-Bernard 2009 Adv. Physiol Edu. 33rd. Volume (4): 270 pages).

The beneficial effects of Ang II treatment on vasodilator and septic shock are currently being discussed (Khanna, A et al., 2017; Antonucci, E. et al., 2017, Tumlin, J.A. et al., 2018). Treatment of patients with vasodilatory shock (80% of septic shock) with angiotensin II was more likely to survive up to 28 days and was significantly associated with hypotension (Khanna, A. Et al., 2017; Tumlin, J.A. et al., 2018)

It is speculated that in patients with shock, angiotensin converting enzyme (ACE) regulation is significantly disturbed, leading to altered angiotensin I / II ratios, and infusion of angiotensin III can compensate for such dysregulation (Tumlin). , J.A. et al., 2018).

Recently, two assays have been devised, characterized and validated specifically for the detection of DPP3 in human body fluids (eg blood, plasma, serum). That is, a fluorescent immunoassay (LIA) that measures the concentration of DPP3 protein and an enzyme capture activity assay (ECA) that detects specific DPP3 activity (Rehfeld et al., 2018 JALM printing). With either method, DPP3 protein or DPP3 activity is actually detected after all disturbing substances have been removed in the washing process. Both methods are so specific that DPP3 can be detected reproducibly in blood samples.

The subject of the present invention is angiotensin receptor agonists and / or precursors thereof for use in the treatment of diseases in a subject.
-The diseases include heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial disease, viral disease (eg AIDS), parasite disease (eg AIDS)). Selected from groups including malaria))), SIRS or septicemia, cancer, acute renal failure (AKI), CNS disorders (eg, seizures, neurodegenerative disorders), autoimmune disorders, vascular disorders, hypotension, and shock.
The subject has an amount and / or DPP3 activity of DPP3 protein in a body fluid sample that exceeds a predetermined threshold.

Kaplan-Meier survival curves associated with low DPP3 plasma concentrations (<68.6 ng / ml) and high DPP3 plasma concentrations (≥68.6 ng / ml). (A) Relationship between 7-day survival rate and DPP3 plasma concentration in patients with septicemia / septic shock (cutoff value is 68.6 ng / ml); (B) 7-day survival rate and DPP3 in patients with cardiogenic shock Relationship between plasma concentration (cutoff value is 68.6 ng / ml); (C) Relationship between 7-day survival rate and DPP3 plasma concentration in patients with acute myocardial infarction (cutoff value is 68.6 ng / ml); (D) Respiration Relationship between 3-month survival rate and DPP3 plasma concentration in patients with difficulty; (E) Relationship between 4-week survival rate and DPP3 plasma concentration in burned patients; (F) 7-day survival rate and DPP3 plasma concentration in patients with septic shock connection of. SDS-PAGE on a gradient gel (4-20%) for natural hDPP3 purified from human erythrocyte lysate. Molecular weight markers are indicated by arrows. Experimental design-Effects of natural hDPP3 in animal models. (A) Injection of DPP3 causes a decrease in the inner diameter shortening rate, which leads to deterioration of cardiac function. (B) Decreased renal function is also observed through the increased renal resistance index. Association-dissociation curve of AK1967-DPP3 binding analysis using Octet. Biosensors loaded with AK1967 were immersed in GST-tagged recombinant human DPP3 serial dilutions (100, 33.3, 11.1, 3.7 nM) to monitor dissociation and association. Western blotting of diluted blood cell lysate and detection of DPP3 using AK1967 as the primary antibody. Suppression curve in which natural DPP3 from blood cells was suppressed using the inhibitory antibody AK1967. The suppression of DPP3 by specific antibodies was concentration-dependent, with an IC50 of approximately 15 ng / ml when analyzed against 15 ng / ml DPP3. Experimental settings-The effect of procizumab on sepsis-induced heart failure. Procizumab dramatically improves inner diameter shortening rate (A) and mortality rate (B) in mice in which heart failure is induced by sepsis. Experimental settings-After inducing cardiac stress with isoproterenol in mice, treat with procizumab (B) and control (A). In mice in which heart failure was induced by isoproterenol, procizumab improved the inner diameter shortening rate within 1 hour after administration (A) and decreased the renal resistance index within 6 hours (B). Experimental settings-Effect of valsartan in healthy mice injected with DPP3. The reduction in inner diameter shortening rate due to DPP3 is relieved by valsartan treatment. High DPP3 concentration levels 24 hours after admission of septic patients were associated with the worst SOFA score. High cDPP3 plasma levels correlate with organ dysfunction in patients with sepsis. Bar graph of SOFA score obtained from changes in DPP3 level during ICU stay in Adren OSS1. HH: DPP3 greater than median at admission and 24 hours later; HL: greater than median at admission but less than median at 24 hours; LL: at admission and 24 hours later Below median; LH: below median at admission, but greater than median at 24 hours. High levels of cDPP3 levels 24 hours after admission of septic patients were associated with the worst SOFA score per organ. Fluctuating cDPP3 levels from admission to 24 hours after admission for (A) heart, (B) kidney, (C) respiratory, (D) liver, (E) coagulation, and (F) central nervous system SOFA score values (HH: high / high, HL: high / low, LH: low / high, LL: low / low).

Initiation of treatment with angiotensin receptor agonists and / or precursors thereof is indicated when the amount and / or DPP3 activity of the DPP3 protein in the body fluid sample exceeds a predetermined threshold level in the patient.

As the patient, a patient with a fatal illness is possible. This means a patient suffering from a fatal disease, any disease process that causes physiological instability leading to incapacity or death within minutes or hours. Disturbances of the nervous and cardiac respiratory systems generally have the most immediate and lethal effects. Such instability can be reliably detected by deviation from the normal range in simple clinical observations (consciousness level, respiratory rate, heart rate, blood pressure, urine volume, etc.). Such measurements can be summarized in a scoring system for assessing the severity of many common diseases (CRB-65 score for pneumonia, Glasgow score for pancreatitis, etc.) (Frost 2007). British Journal of Hospital Medicine Vol. 68 (10): M180-183).

The diseases include heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial disease, viral disease (eg AIDS), parasite disease (eg malaria). )), SIRS or septicemia, cancer, acute renal failure (AKI), CNS disorder (eg, seizures, neurodegenerative disease), autoimmune disease, vascular disease, hypotension, and shock.

Therefore, shock is possible as the disease, and the disease can be selected from the group including hypovolemia shock, cardiogenic shock, vascular obstructive shock, and distributive shock.

Shock is characterized by hypoxia in cells and tissues due to decreased oxygen supply and / or increased oxygen consumption, or inadequate oxygen utilization. Shock is a fatal condition of circulatory failure, most commonly manifested as hypotension (systolic blood pressure <90 mm Hg, or MAP <65 mm Hg). Shock can be divided into four types based on the underlying cause: hypovolemia, cardiogenic, vascular occlusion, and abnormal blood distribution (Vincent and De Backer 2014 N. Engl. J. Med. Volume 370 (6): 583 pages).

Hypovolemia shock is characterized by a decrease in intravascular volume and can be divided into two broad subtypes. That is, it is bleeding and non-bleeding. Common causes of hemorrhagic hypovolemia are gastrointestinal bleeding, trauma, vascular etiology (eg, ruptured abdominal aortic aneurysm, tumor invasion into major blood vessels), and in the setting of anticoagulant use. Includes spontaneous bleeding. Common causes of non-hemorrhagic hypovolemia are vomiting, diarrhea, loss from the kidneys, loss from the skin / loss of insensitivity (eg burns, heat stroke), or pancreatitis, liver cirrhosis, intestinal obstruction. Includes loss to the stroma in the setting of trauma. For an overview, see Koya and Paul 2018 Shock, StatPearls [Internet], Treasure Island, Florida: StatPearls Publishing; 2019-October 27, 2018.

Cardiogenic shock (CS) is defined as a critical terminal organ hypoperfusion condition due to decreased cardiac output. In particular, CS forms a spectrum ranging from mild hypoperfusion to significant shock. Established criteria for the diagnosis of CS are: (i) systolic blood pressure is less than 90 mmHg over 30 minutes or requires a pressor agent to achieve blood pressure above 90 mmHg; (ii) lungs. Congestion, or increased left ventricular filling pressure; (iii) signs of impaired organ perfusion and the following criteria: (a) altered mental state; (b) cold, sticky skin; (c) oliguria Urine (urine volume <30 ml / hour); (d) with at least one increase in serum lactate (Reynolds and Hochman 2008 Circulation Vol. 117: pp. 686-697). Acute myocardial infarction (AMI) and subsequent ventricular dysfunction are the most common causes of CS, explaining about 80% of the causes. Mechanical complications (such as interventricular septum (4%) and free wall rupture (2%)) and acute severe mitral regurgitation (7%) are less likely to cause CS after AMI (Hochman). 2000 J Am Coll Cardiol Vol. 36: pp. 1063-1070). Non-AMI-related CS can be caused by decompensated valvular heart disease, acute cardiomyopathy, arrhythmia, etc., and there are multiple treatment options.

Vascular occlusion shock is due to the physical obstruction of a large blood vessel or the heart itself. This form of shock can result from several conditions (eg, cardiac tamponade, tension pneumothorax, pulmonary embolism, aortic stenosis). For an overview, see Koya and Paul 2018 Shock, StatPearls [Internet], Treasure Island, Florida: StatPearls Publishing; October 27, 2019-October 27, 2018.

There are four types of distributive shock depending on the cause. That is, neurogenic shock (reduced sympathetic nerve stimulation and reduced vascular tone), anaphylactic shock, septic shock, and shock due to adrenal crisis. Distributive shock can be caused by systemic inflammatory response syndrome (SIRS) due to non-infectious causes (pancreatitis, burns, trauma, etc.) in addition to sepsis. Other causes include toxin shock syndrome (TSS), anaphylaxis (sudden severe allergic reaction), adrenal insufficiency (acute exacerbation of chronic adrenal insufficiency, destruction or elimination of adrenal glands, and foreign steroids. Adrenal gland depression, hypopituitarism, and dysmetabolism of the hormones produced), response to drugs or toxins, heavy metal poisoning, hepatic insufficiency, and damage to the nervous system. For an overview, see Koya and Paul 2018 Shock, StatPearls [Internet], Treasure Island, Florida: StatPearls Publishing; 2019-October 27, 2018.

In one embodiment of the invention, the patient is a shock patient. In yet another embodiment of the invention, the shock can be selected from the group of blood volume reducing shock, cardiogenic shock, vascular occlusion shock, and distributive shock. In another particular embodiment of the invention, the distributive shock is septic shock.

Agonists are chemical substances that bind to a receptor and activate the receptor, causing a biological reaction. Receptors can be activated by endogenous agonists (such as hormones and neurotransmitters) or exogenous agonists (such as drugs), resulting in a biological response. A fully agonist binds to a receptor and activates the receptor with the greatest response that the agonist can elicit at the location of the receptor. A partial agonist is a drug that binds to a given receptor and activates that receptor, but has only a partial effect at the position of that receptor compared to a full agonist. Efficacy is the amount of agonist required to elicit the desired reaction. Agonist efficacy is inversely related to its _EC50 value. EC ₅₀ can be measured for a given agonist by determining the concentration of agonist required to induce half of the maximum biological response of that agonist. _EC50 values are useful for comparing the efficacy of drugs that have similar effects and produce similar physiological effects. The smaller the _EC50 value, the greater the efficacy of the agonist and the lower the concentration of drug required to elicit the maximum biological response.

Treatment with angiotensin receptor agonists and / or precursors thereof can be continued as long as the patient has the amount and / or DPP3 activity of DPP3 protein in the body fluid sample above a predetermined threshold level.

The amount and / or DPP3 activity of the DPP3 protein is at least every 24 hours, preferably every 12 hours, more preferably every 8 hours, even more preferably every 6 hours, even more preferably every 4 hours. It is even more preferably measured every 2 hours, even more preferably every hour, and most preferably every 30 minutes.

Treatment with angiotensin receptor agonists and / or precursors thereof can be discontinued when the patient has an amount and / or DPP3 activity of DPP3 protein in the fluid sample that is below a predetermined threshold level.

In one embodiment, the angiotensin receptor agonist and / or precursor thereof is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, and angiotensin IV.

Angiotensin remedy:

Angiotensin I, also called proangiotensin, is formed by the action of renin on angiotensinogen. Renin cleaves the peptide bond between the leucine (Leu) residue and the valine (Val) residue, giving rise to the decapeptide (10 amino acids) (des-Asp) angiotensin I. Angiotensin I has two C-terminal residues removed by angiotensin converting enzyme (ACE) (mainly ACE in the lungs (but endothelial cells, renal epithelial cells, and ACE is also present in the brain)). By doing so, it is converted to angiotensin II.

Angiotensin II, angiotensin III, and angiotensin IV are peptide hormones naturally produced by the body that regulate blood pressure through vasoconstriction and sodium reabsorption. The hemodynamic effects of angiotensin II administration have been the subject of numerous clinical studies and have demonstrated significant effects on systemic and renal blood flow (Harrison-Bernard, L.M., "Kidney renin-angiotensin system" Adv Physiol. Educ, Volume 33 (4): 270 pages (2009)).

Angiotensin II is a hormone produced by the renin-angiotensin-aldosterone system (RAAS) that regulates blood pressure through regulation of vascular smooth muscle tone and extracellular fluid homeostasis. Angiotensin II is the target of many hypertension treatments because it conveys its effects on the vascular system by inducing vasoconstriction and sodium retention. In addition to its systemic effect, angiotensin II has a prominent effect on the efferent arterioles of the kidney and maintains glomerular filtration when blood flow is reduced. Angiotensin II also regulates renal reabsorption by stimulating Na ⁺ / H ⁺ exchange in the proximal tubule and inducing the release of aldosterone and vasopressin (Harrison-Bernard, LM, "Kidney renin-angiotensin". System "Adv Physiol Educ, Volume 33 (4): 270 pages (2009)).

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 13) is possible as an angiotensin II therapeutic agent that can be used with the compositions and methods of the present disclosure, which is 5-isoleucine angiotensin II. Also called. SEQ ID NO: 13 is an octapeptide that is naturally present in humans and other species (horses, pigs, etc.). Isoleucine can be replaced with valine, resulting in 5-valine angiotensin II, i.e. Asp-Arg-Val-Tyr-Val-His-Pro-Phe (SEQ ID NO: 14). Other angiotensin II analogs, such as [Asn ¹ -Phe ⁴ ]-angiotensin II (SEQ ID NO: 15), hexapeptide Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 16), nonapeptide Asn-Arg-Val- Tyr-Tyr-Val-His-Pro-Phe (SEQ ID NO: 17), [Asn ¹ Ile ⁵ Ile ⁸ ]-Angiotensin II (SEQ ID NO: 18), [Asn ¹ -lle ⁵ -Ala ⁸ ]-Angiotensin II (SEQ ID NO: 18) 19), and [Asn ¹ -Gindo Tyr ⁴ -Ile ⁵ -Angiotensin II (SEQ ID NO: 20) can also be used. Angiotensin II can be synthesized, for example, by incorporating a modification such as C-terminal amidation by solid phase peptide synthesis. The term "angiotensin II" shall mean any of these various forms, as well as any combination thereof, unless further specified.

In some aspects, compositions comprising angiotensin II are 5-valin angiotensin III, 5-valin angiotensin III amide, 5-L-isoleucine angiotensin III, and 5-L-isoleucine angiotensin III amide, or as pharmaceuticals thereof. You can choose from acceptable salts, which are preferably manufactured under current Proper Manufacturing Standards (cGMP). In some aspects, the composition can contain different forms of angiotensin II in different proportions (eg, a mixture of hexapeptide angiotensin II and nonapeptide angiotensin II). Compositions containing angiotensin II may be suitable for parenteral administration (eg injection or intravenous infusion).

The sequence of angiotensin II used in the compositions and methods disclosed herein can be homologous to the sequence of angiotensin II described above. In some aspects, to the present invention, in isolated amino acid sequences, synthetic amino acid sequences, or recombinant amino acid sequences, SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, and / or 20 and at least 80. Includes%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% matches. Any such variant sequence can be used in place of the angiotensin II described in the previous paragraph.

For all sequences, sequence matching is determined according to the following method. That is, percent match is calculated by multiplying the number of matches in a pair by 100 and dividing by the length of the aligned area (including the gap) (percent match = [number of matches x 100] / alignment). Area length (including gaps).

Angiotensin III is a metabolite of angiotensin II and has about 40% activity of angiotensin II. Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 21) is possible as an angiotensin III therapeutic agent that can be used with the compositions and methods of the present disclosure. SEQ ID NO: 21 is a heptapeptide that is naturally present in humans and other species (horses, pigs, etc.). Isoleucine can be replaced with valine, resulting in Arg-Val-Tyr-Val-His-Pro-Phe (SEQ ID NO: 22). Other angiotensin III analogs, such as [Phe ³ ]-angiotensin III (SEQ ID NO: 23), [Ile ⁴ -Ala ⁷ ]-angiotensin III (SEQ ID NO: 24), and [Gindo Tyr ³ -Ile ⁴ ]-angiotensin III ( SEQ ID NO: 25) can also be used. Angiotensin III can be synthesized, for example, by incorporating a modification such as C-terminal amidation by solid phase peptide synthesis. The term "angiotensin III" shall mean any of these various forms, as well as any combination thereof, unless further specified.

In some aspects, compositions comprising angiotensin III can be used as 4-valin angiotensin III, 4-valin angiotensin III amide, 4-L-isoleucine angiotensin III, and 4-L-isoleucine angiotensin III amide, or pharmaceuticals thereof. You can choose from acceptable salts, which are preferably manufactured under current Proper Manufacturing Standards (cGMP). Compositions containing angiotensin III may be suitable for parenteral administration (eg injection or intravenous infusion).

The sequence of angiotensin III used in the compositions and methods disclosed herein can be homologous to the sequence of angiotensin III described above. In some aspects, to the present invention, in isolated amino acid sequences, synthetic amino acid sequences, or recombinant amino acid sequences, SEQ ID NOs: 21, 22, 23, 24, and / or 25 and at least 80%, 85%, 90. Includes%, 95%, 97%, 98%, 99%, or 100% matches. Any such variant sequence can be used in place of the angiotensin II described in the previous paragraph.

Angiotensin IV is a metabolite of angiotensin III and has less activity than angiotensin II. Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 26) is possible as an angiotensin IV therapeutic agent that can be used with the compositions and methods of the present disclosure. SEQ ID NO: 26 is a hexapeptide that is naturally present in humans and other species. Isoleucine can be replaced with valine, resulting in Val-Tyr-Val-His-Pro-Phe (SEQ ID NO: 27). Other angiotensin IV analogs, such as [Phe ² ]-angiotensin III (SEQ ID NO: 28), [Ile ³ -AIa ⁶ ]-angiotensin IV (SEQ ID NO: 29), and [Gindo Tyr ² -Ile ³ ]-angiotensin IV ( SEQ ID NO: 30) can also be used. Angiotensin IV can be synthesized, for example, by incorporating a modification such as C-terminal amidation by solid phase peptide synthesis. The term "angiotensin IV" shall mean any of these various forms, as well as any combination thereof, unless further specified.

In some aspects, compositions comprising angiotensin IV can be used as 3-valine angiotensin IV, 3-valine angiotensin IV amide, 3-L-isoleucine angiotensin IV, and 3-L-isoleucine angiotensin IV amide, or pharmaceuticals thereof. You can choose from acceptable salts, which are preferably manufactured under current Proper Production Standards (cGMP). Compositions containing angiotensin IV may be suitable for parenteral administration (eg injection or intravenous infusion).

The sequence of angiotensin IV used in the compositions and methods disclosed herein can be homologous to the sequence of angiotensin IV described above. In some aspects, to the present invention, in an isolated amino acid sequence, a synthetic amino acid sequence, or a recombinant amino acid sequence, SEQ ID NO: 25, 26, 27, 28, 29, and / or 30 and at least 80%, 85%. , 90%, 95%, 97%, 98%, 99%, or 100% match. Any such variant sequence can be used in place of the angiotensin IV described in the previous paragraph.

Angiotensin II therapeutic agents, angiotensin III therapeutic agents, or angiotensin IV therapeutic agents are disclosed in the above peptides, among them United States Patent Application Publication No. 2011/0081371 (incorporated herein by reference). Any suitable salt (eg, acetate) of a peptide or conjugate (including PEGylated form, as such), unprotected form, acetylated form, deacetylated form, And / or can be used in the form of prodrugs. The term "prodrug" means a precursor compound capable of producing or releasing the above peptides under physiological conditions. Larger peptides that are selectively cleaved to form the peptides of the invention are possible as such prodrugs or precursors. For example, in some aspects, prodrugs or precursors can be angiotensinogen, angiotensin I, or homologues thereof that become angiotensin II by the action of some endogenous or exogenous enzymes. Included in additional prodrugs or precursors are peptides with protected amino acids having, for example, one or more carboxylic acid groups and / or protecting groups at the positions of amino groups. Suitable protecting groups for amino groups include benzyloxycarbonyl group, t-butyloxycarbonyl (BOC) group, fluorenylmethyloxycarbonyl (FMOC) group, formyl group, acetyl group, acyl group. Either. Suitable protecting groups for carboxylic acid groups include esters such as benzyl esters and t-butyl esters. In the present invention, amino acid substitutions, deletions, additions (substitutions and additions include standard D and L amino acids), and modifications with angiotensin II, angiotensin III, angiotensin IV, and / or precursor peptides. It is also considered to use those that have amino acids (eg, amidated amino acids and acetylated amino acids), but whose underlying peptide sequence therapeutic activity is maintained at pharmacologically useful levels.

In a preferred embodiment, the angiotensin II is angiotensin II acetate. The angiotensin II acetate is L-aspartyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalanine acetate. Counterion acetates are present in non-stoichiometric ratios. The molecular formula of angiotensin II acetate is C ₅₀ H ₇₁ N ₁₃ O ₁₂ · (C ₂ H ₄ O ₂ ) _n (n is the number of acetate molecules; theoretical n = 3), and the average molecular weight is 1046.2. (As a free base).

One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, wherein the subject is the amount and / or amount of DPP3 protein in a body fluid sample that exceeds a predetermined threshold. Having DPP3 activity, the amount and / or DPP3 activity of said DPP3 protein is at least 1 in the body fluid sample of said subject before and / or during treatment with said angiotensin receptor agonist and / or precursor thereof. It has been decided once.

One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, wherein the subject is the amount and / or amount of DPP3 protein in a body fluid sample that exceeds a predetermined threshold. The amount of DPP3 protein and / or the determination of DPP3 activity determined in the body fluid sample having DPP3 activity is used as a treatment guide and / or treatment monitoring for said treatment with angiotensin receptor agonists and / or precursors thereof. ..

In one embodiment of the invention, the amount and / or DPP3 activity of DPP3 protein in a body fluid sample is determined prior to treatment of the subject with angiotensin receptor agonists and / or precursors thereof. In one embodiment of the invention, the amount and / or DPP3 activity of the DPP3 protein in the body fluid sample is at least once, preferably at least twice, or during treatment of the subject with angiotensin receptor agonist and / or precursor thereof. It is preferably determined at least once daily during treatment. In one embodiment of the invention, the amount and / or DPP3 activity of the DPP3 protein in the body fluid sample is determined after treatment of the subject with an angiotensin receptor agonist and / or precursor thereof. In one embodiment of the invention, the amount and / or DPP3 activity of the DPP3 protein in the body fluid sample is pre-treatment and / or during and / or post-treatment of the subject with angiotensin receptor agonist and / or precursor thereof. It is determined.

One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, wherein the subject is the amount and / or amount of DPP3 protein in a body fluid sample that exceeds a predetermined threshold. The body fluid sample having DPP3 activity and the subject is selected from whole blood, plasma, and serum.

One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, wherein the angiotensin receptor agonist and / or precursor thereof is administered to the subject in a pharmaceutical formulation. To.

One embodiment of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject, wherein the angiotensin receptor agonist and / or a precursor thereof is angiotensin II, the pharmaceutical preparation. Is a solution, preferably a solution that can be used as is. In another embodiment, the subject matter of the invention is further a pharmaceutical product according to the invention, wherein the pharmaceutical product is in a dry state reconstituted prior to use.

The pharmaceutical compositions of the present invention can also contain diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "medically acceptable base" is a non-toxic substance that can be administered to a patient together with a therapeutically effective substance of the invention (such as angiotensin II) and does not destroy the pharmacological activity of the therapeutically effective substance. Means the base. The expression "medically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient.

The characteristics of the base will depend on the route of administration. The term "excipient" means an additive in a formulation or composition that is not a pharmaceutically active ingredient.

Those of skill in the art will recognize that the choice of any one excipient can influence the choice of any other excipient. For example, selecting one particular excipient may prevent the use of one or more additional excipients. This is because the combination of excipients can produce undesired effects. Non-limiting examples of excipients of the invention include cosolvents, solubilizers, buffers, pH regulators, bulking agents, surfactants, encapsulants, tonicity regulators, stabilizers, protections. Agent and viscosity modifier.

In some aspects, it may be advantageous to include a pharmaceutically acceptable base in the composition of the invention.

In some aspects, it may be advantageous to include the solubilizer in the composition of the invention. Solubils are useful for increasing the solubility of any component of a formulation or composition, including therapeutically effective substances (eg, angiotensin II, angiotensin III, angiotensin IV) or excipients. there is a possibility. The solubilizers described herein are not intended to constitute an exhaustive list, but merely provide a representative solubilizer that can be used in the compositions of the present invention. In some respects, non-limiting examples of solubilizers include ethyl alcohol, t-butyl alcohol, polyethylene glycol, glycerol, methylparaben, propylparaben, polyvinylpyrrolidone, and any pharmaceutically acceptable of these. Salt and / or combination of.

In some aspects, it may be advantageous to regulate the pH of a composition by including a pH regulator in the composition of the invention. Changing the pH of a formulation or composition has, for example, a beneficial effect on the stability or solubility of a therapeutically effective substance, or a beneficial effect in producing a formulation or composition suitable for parenteral administration. There may be. pH regulators are well known in the art. Accordingly, the pH regulators described herein are not intended to constitute an exhaustive list, but merely provide a representative pH regulator that can be used in the compositions of the present invention. Acids and bases can be included in pH regulators, for example. In some respects, non-limiting examples of pH regulators include acetic acid, hydrochloric acid, phosphoric acid, sodium hydroxide, sodium carbonate, and combinations thereof.

The pH of the composition of the invention can be any pH that provides the desired properties for the formulation or composition. Desirable properties include, for example, the stability of therapeutically effective substances (eg, angiotensin II, angiotensin III, or angiotensin IV), retention of therapeutically effective substances when compared to compositions at other pH values. Increased sex and improved filtration efficiency. In some aspects, the pH of the composition of the invention can be from about 3.0 to about 9.0, such as from about 5.0 to about 7.0. In a particular aspect, the pH of the composition of the invention is 5.5 ± 0.1, 5.6 ± 0.1, 5.7 ± 0.1, 5.8 ± 0.1, 5.9 ± 0.1, 6.0 ± 0.1, 6.1 ± 0.1, 6.2 ± 0.1, 6.3 ± 0.1, 6.4 ± 0.1 or 6.5 ± 0.1 is possible.

In some aspects, it may be advantageous to contain one or more buffers in the composition to reduce pH fluctuations. In some aspects, the buffer can have, for example, about 5.5, about 6.0, or about 6.5 pKa. Those skilled in the art will recognize that suitable buffers can be selected and included in the compositions of the invention based on pKa and other properties.

Buffers are well known in the art. Accordingly, the buffers described herein are not intended to form an exhaustive list, but merely provide a representative buffer that can be used in the compositions of the present invention. On some aspects, the buffer can include Tris, Tris HCl, potassium phosphate, sodium phosphate, sodium citrate, sodium ascorbate, a combination of sodium phosphate and potassium phosphate, Tris / Tris HCl. , Sodium hydrogen carbonate, arginine phosphate, arginine hydrochloride, histidine hydrochloride, cacodylate, succinate, 2- (N-morpholino) ethanesulfonic acid (MES), maleate, bis-tris, phosphate , One or more of the carbonates, and any pharmaceutically acceptable salt and / or combination thereof.

In some aspects, it may be advantageous to include a surfactant in the composition of the invention. Surfactants generally reduce the surface tension of liquid compositions. Surfactants may provide advantageous properties such as improved ease of filtration. Surfactants may also function as emulsifiers and / or solubilizers. Surfactants are well known in the art. Accordingly, the surfactants described herein are not intended to constitute an exhaustive list, but merely provide a representative surfactant that can be used in the compositions of the present invention. Non-limiting examples of surfactants that can be included include sorbitan esters (such as polysorbate (eg, polysorbate 20 and polysorbate 80)), lipopolysaccharides, polyethylene glycol (eg, PEG 400 and PEG 3000), poloxamers (eg, PEG 400 and PEG 3000). Pluronic), ethylene oxide and polyethylene oxide (eg Triton X-100), saponin, phospholipids (eg recithin), and combinations thereof.

In some aspects, it may be advantageous to include a tonicity adjuster in the composition of the invention. Tonicity of the liquid composition is one important consideration when administering the composition to the patient, for example by parenteral administration. Therefore, a tonicity adjuster can be used to help make a suitable formulation or composition for administration. Tonicity adjusters are well known in the art. Accordingly, the tonicity adjusters described herein are not intended to constitute an exhaustive list, but merely provide a representative tonicity adjuster that can be used in the compositions of the present invention. .. Tonicity adjusters can be ionic or non-ionic, and non-limiting examples thereof include inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates. Typical inorganic salts can include sodium chloride, potassium chloride, sodium sulphate, and potassium sulphate. One typical amino acid is glycine. Typical sugars can include sugar alcohols (such as glycerol), propylene glycol, glucose, sucrose, lactose, and mannitol.

In some aspects, it may be advantageous to include stabilizers in the compositions of the invention. Stabilizers help increase the stability of therapeutically effective substances contained in the compositions of the present invention. This can occur, for example, by reducing the degradation of therapeutically effective substances or by blocking the aggregation of therapeutically effective substances. Without wishing to be bound by theory, mechanisms that increase stability may include blocking therapeutically effective substances from solvents or suppressing free radical oxidation of anthracycline compounds. Stabilizers are well known in the art. Therefore, the stabilizers described herein are not intended to constitute an exhaustive list, but merely provide representative stabilizers that can be used in the compositions of the present invention. Non-limiting examples of stabilizers include emulsifiers and surfactants.

The composition of the present invention can be administered in a variety of conventional ways. In some aspects, the compositions of the invention are suitable for parenteral administration. These compositions can be administered, for example, intraperitoneally, intravenously, intrarenalally, or intrathecally. In some aspects, the compositions of the invention are injected intravenously. Those skilled in the art will appreciate that the method of administering a formulation or composition of a therapeutically effective substance according to the invention depends on factors such as the age, weight, and physical condition of the patient being treated, and the disease or condition being treated. You will be aware that you will be. Therefore, those skilled in the art will be able to select the most suitable administration method for the patient on a case-by-case basis.

Angiotensin receptor agonists and / or precursors thereof can be administered in any suitable manner, but are typically administered by continuous infusion. Therefore, the increase or decrease of the administration rate can be realized by changing the flow rate of the intravenous drip, changing the concentration of the drug contained in the intravenous drip, and the like. However, the method of varying the rate of administration will depend on the method of administration of the therapeutic agent. If the therapeutic agent is administered transmucosally or transdermally, the rate can be increased, for example by changing to a patch or transdermal composition that is released at a higher rate. If the therapeutic agent is orally administered, the rate may be increased, for example, by switching to a higher dose form, administering an additional dose, or administering a controlled release form with a higher release rate. can.

If the therapeutic agent is administered by inhalation, the rate can be increased, for example, by administering an additional bolus, a more concentrated bolus, or a faster-release bolus. Other dosing regimens (through subcutaneous infusion pumps, suppositories, etc.) can be modified in the same way, reducing the dosing rate by performing the reverse of what is believed to increase the dosing rate of the therapeutic agent. can.

One embodiment of the present invention is an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject, wherein the angiotensin receptor agonist and / or a precursor thereof is 0.1 to 200 ng / kg /. Minutes, preferably 1-100 ng / kg / min, more preferably 2-80 ng / kg / min, even more preferably 5-60 ng / kg / min, even more preferably 10-50 ng / kg / min. Angiotensin II, even more preferably administered at a rate of 15-40 ng / kg / min, most preferably at a rate of 20 ng / kg / min.

In one particular embodiment of the invention, the starting dose (initial rate) of angiotensin II is 80 ng / kg / min, more preferably 40 ng / kg / min, most preferably 20 ng / min by continuous intravenous infusion. kg / min.

In another particular embodiment of the invention for titrating angiotensin II, the blood pressure response (eg, mean arterial pressure; MAP) is monitored. Titration of angiotensin II is performed every 60 minutes, more preferably every 45 minutes, even more preferably every 30 minutes, even more preferably every 15 minutes, even more preferably every 10 minutes, and most preferably every 5 minutes. be able to. Titration of angiotensin II is performed up to 40 ng / kg / min, more preferably up to 20 ng / kg / min, most preferably 15 ng / kg / min, as needed to achieve or maintain the targeted blood pressure. It can be carried out while gradually increasing. In another preferred embodiment, the dose of angiotensin II should not exceed 80 ng / kg / min for the first 3 hours of treatment. It is most preferable that the maintenance dose does not exceed 40 ng / kg / min and that a low dose of 1.25 ng / kg / min can be used.

In some embodiments, the patient has an initial mean arterial pressure (MAP) of about 40 mm Hg, about 45 mm Hg, about 50 mm Hg, about 55 mm Hg, about 60 mm Hg before administering the composition. , About 65 mm Hg, about 70 mm Hg, or about 75 mm Hg. The treatment method measures the mean arterial pressure of the patient, and the mean arterial pressure is about 40 mm Hg, about 45 mm Hg, about 50 mm Hg, about 55 mm Hg, about 60 mm Hg, about 65 mm Hg, about. Increasing the dosing rate of angiotensin II can be included if it is below 70 mm Hg, or about 75 mm Hg.

In one embodiment of the invention, the patient can be administered a pressor agent (eg, catecholamines such as norepinephrine, norepinephrine equivalents, epinephrine, dopamine, phenyleffrin), or a combination thereof. In some embodiments, the pressor agent is vasopressin (telripressin, argypressin, desmopressin, ferripressin, repressin, or ornipressin).

One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, wherein the angiotensin receptor agonist and / or precursor thereof is particularly angiotensin II, DPP3. Administered in combination with an inhibitor.

One embodiment of the invention is a combination of an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject and an inhibitor of DPP3, wherein the inhibitor of DPP3 is an anti-DPP3 antibody or anti-DPP3. Selected from the group containing antibody fragments or anti-DPP3 non-Ig scaffolds.

According to the present invention, the "anti-DPP3 antibody" is an antibody that specifically binds to DPP3, the "anti-DPP3 antibody fragment" is a fragment of the anti-DPP3 antibody, and the fragment specifically binds to DPP3. do. An "anti-DPP3 non-Ig scaffold" is a non-Ig scaffold that specifically binds to DPP3.

One embodiment of the invention is a combination of an angiotensin receptor agonist and / or a precursor thereof and an inhibitor of DPP3 for use in the treatment of a disease in a subject, wherein the inhibitor of DPP3 is SEQ ID NO: 1, in particular sequence. An anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to number 2.

One embodiment of the present invention is a combination of an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject and an inhibitor of DPP3, wherein the inhibitor of DPP3 is ^10-7 M or less. An antibody or fragment or scaffold that exhibits the lowest binding affinity for DPP3.

According to the invention, one of ordinary skill in the art is well aware that the binding affinity of the DPP3 binder disclosed herein to DPP3 can be measured by a variety of suitable assays known in the art. Representative examples are shown below, but these examples should not be construed as limiting the possibility of measuring the binding affinity of the DPP3 binder disclosed herein for DPP3.

For example, the binding affinity of the DPP3 binder for an epitope can be determined. Binding assays can be performed, for example, to detect and / or quantify antibodies that bind to the immunized peptide of each binder. For example, this immunized peptide can be immobilized on the surface of a solid phase. A test sample (eg, an antibody solution) can be passed over an immunized peptide that does not move and the bound antibody can be detected. For purposes herein, the term "solid phase" can include any material or vessel in which the assay can be performed internally or on the surface, and includes non-limiting examples of solid phases. , Porous materials, non-porous materials, test tubes, wells, slides, magnetic beads, etc.

Typical detection methods are as follows:
—— Label the antibody prior to contact with the solid phase and detect each label (fluorescent label, chemiluminescent label, enzyme label, etc.);
--Sample-Use a labeled secondary antibody for the specific Fc portion of the antibody. Antibodies bound to the solid phase are incubated with secondary antibodies (eg, anti-human IgG, anti-mouse IgG) to detect each label (fluorescent label, chemiluminescent label, enzyme label, etc.);
--Use a labeled antibody (eg labeled AK1967) as a competitor for binding to the solid phase;
--Quantify the binding affinity by reducing the signal.

As another method for determining the affinity of the antibody for DPP3, the dynamics of the binding of DPP3 to the immobilized antibody was measured on an unlabeled surface using the Biacore 2000 series (GE Healthcare Europe GmbH, Freiburg, Germany). It can be obtained by plasmon resonance. Reversible immobilization of the antibody can be performed using anti-mouse Fc antibodies covalently bound to the surface of the CM5 sensor (mouse antibody capture kit; GE Healthcare) (Lorenz et al., 2011 Antimicrob Agents Chemother). Volume 55 (1): pp. 165-173).

One embodiment of the invention is a combination of an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject and an inhibitor of DPP3, wherein the inhibitor of DPP3 is unispecific. An antibody or fragment or scaffold. In one embodiment, the inhibitor of DPP3 is a monoclonal antibody or fragment or scaffold.

Unispecific antibodies or fragments or non-Ig scaffolds according to the invention are antibodies or fragments or non-Ig scaffolds that all have an affinity for the same antigen. Monoclonal antibodies are unispecific, but unispecific antibodies can also be produced by means other than those produced from one common germ cell.

In one particular embodiment, the capture binder bound to full-length DPP3 specifically suppresses DPP3 activity by less than 50%, preferably less than 40%, more preferably 30% in a liquid phase assay. See the description above for the definition of a liquid phase assay. In one particular embodiment that blocks the suppression of DPP3, the capture binder shall not bind to DPP3 in the region around the active center and substrate binding region (amino acids 316-669 of SEQ ID NO: 1).

One embodiment of the invention is a combination of an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject and an inhibitor of DPP3, wherein the inhibitor of DPP3 is an antibody or fragment or scaffold. There is full-length DPP3 binding and DPP3 activity of at least 10%, or at least 50%, more preferably at least 60%, even more preferably greater than 70%, even more preferably greater than 80%, even more preferably 90. %, And even more preferably> 95%.

Suppression of DPP3 activity by a binder in a liquid phase assay can be determined as follows: In a liquid phase assay, possible DPP3 capture binders are incubated with recombinant DPP3 or purified native DPP3 and specific DPP3 substrates. It is preferable to select the capture binder for ECA that has the smallest suppression capacity. The capture binder should suppress DPP3 activity by less than 50%, preferably less than 40%, preferably less than 30%. A special liquid phase DPP3 activity assay to determine the ability to suppress possible DPP3 capture binders involves the following steps:
• Incubate 25 ng / ml natural human DPP 3 with 5 μg / ml capture binder and buffer control in 50 mM Tris-HCl, pH 7.5 and 100 μM ZnCl ₂ for 1 hour at room temperature.
-Add the fluorescent substrate Arg-Arg-βNA (20 μl, 2 mM).
• Incubate at 37 ° C. and monitor free βNA production in a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH) for 1 hour. Fluorescence of βNA is detected by exciting at 340 nm and measuring emission at 410 nm.
-Calculate the gradient (unit: RFU / min) in which the fluorescence of different samples increases. A gradient of natural human DPP3 using a buffer control is 100% active. A possible capture inhibitor inhibitory activity is defined as a reduction in natural human DPP3 activity (in%) by incubation with the capture binder.

In the liquid phase assay, the body fluid sample is directly exposed to a fluorescent substrate (eg Arg-Arg-β-NA). Due to the presence of many different aminopeptidases in plasma (Sanderink et al., 1988), the substrate used is cleaved by peptidases other than DPP3. To avoid this problem, one preferred method for detecting specific DPP3 activity is the use of an enzyme capture activity assay.

In one particular embodiment, the method for determining active DPP3 in an enzyme capture assay comprises the following steps:
-The step of contacting the sample with a capture binder that binds to full-length DPP3 but preferably suppresses DPP3 activity by less than 50%, preferably less than 40%, more preferably 30% in a liquid phase assay. To prevent suppression of DPP3, the capture binder must not bind to DPP3 in the active center and the region around the substrate binding region (amino acids 316-669 of SEQ ID NO: 1).
-Step of separating DPP3 bound to the capture binder from the body fluid sample,
-Step of adding the substrate of DPP3 to the separated DPP3,
-The step of quantifying DPP3 activity by measuring the conversion of the substrate of DPP3.

In the present invention, an "antibody" is a protein (including one or more polypeptides) substantially encoded by an immunoglobulin gene that specifically binds to an antigen. Known immunoglobulin genes include kappa, lambda, alpha (IgA), gamma (IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ ), delta (IgD), epsilon (IgE), and mu (IgE). In addition to the constant region gene of IgM), it is a large number of immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally about 25 kDa, or 214 amino acids in length.

Full-length immunoglobulin heavy chains are generally about 50 kDa, or 446 amino acids in length. The light chain is encoded by a variable region gene at the NH ₂ terminal (about 110 amino acids in length) and a kappa constant region gene or a lambda constant region gene at the COOH terminal. Heavy chains are also encoded by a variable region gene (approximately 116 amino acids in length) and one of the other constant region genes.

The basic structural unit of an antibody is generally a tetramer consisting of the same two immunoglobulin chain pairs, each pair having one light chain and one heavy chain. In each pair, the light chain variable region and the heavy chain variable region bind to the antigen, and the constant region mediates the effector function. Immunoglobulins are also present in a variety of other forms, such as Fv, Fab, and F (ab') ₂ , as well as bifunctional hybrid and single-chain antibodies (eg, Lanzavecchia et al., Eur. J). Immunol. Volume 17: 105 pages, 1987; Huston et al., Proc. Natl. Acad. Sci. USA Volume 85: 5879-5883, 1988; Bird et al., Science Volume 242: 423-426, 1988; Hood et al., "Immunology", Benjamin, New York, 2nd Edition, 1984; Hunkapiller and Hood, Nature Vol. 323: pp. 15-16, 1986).

The light chain or heavy chain variable region of an immunoglobulin contains one framework region divided by three hypervariable regions (also called complementarity determining regions (CDRs)) ("Immunologically of interest". See Sequences of Certain Proteins, E. Kabat et al., United States Ministry of Health and Welfare, 1983). As pointed out above, CDRs are primarily responsible for the binding of antigens to epitopes. An immune complex is an antibody that specifically binds to an antigen (eg, a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody) or a functional antibody fragment.

A "chimeric antibody" is an antibody composed of a variable region gene and a constant region gene of an immunoglobulin in which a light chain gene and a heavy chain gene typically belong to different species by genetic manipulation. For example, a variable compartment of a gene from a mouse monoclonal antibody can be conjugated to a human constant compartment (such as kappa and gamma 1 or gamma 3). In one example, a therapeutic chimeric antibody is therefore a hybrid protein consisting of a variable domain or antigen binding domain from a mouse antibody and a constant domain or effect domain from a human antibody, but using other mammalian species or variable regions. It can be produced by molecular technology. Methods for making chimeric antibodies are well known in the art, see, for example, US Pat. No. 5,807,715. A "humanized" immunoglobulin is an immunoglobulin containing one or more CDRs from the human framework region and non-human (eg, mouse, rat, or synthetic) immunoglobulins. The non-human immunoglobulin that provides the CDR is referred to as the "donor" and the human immunoglobulin that provides the framework is referred to as the "acceptor".

In one embodiment of the invention, all CDRs in humanized immunoglobulins are derived from donor immunoglobulins. The constant region may not be present, but if it is, it must be substantially the same as the human immunoglobulin constant region, i.e. at least about 85-90% (eg, about 95% or more). .. Thus, humanized immunoglobulins are probably all but substantially the same as the corresponding parts of the native humanized immunoglobulin sequence.

The "humanized antibody" according to the present invention is an antibody containing a humanized light chain immunoglobulin and a humanized heavy chain immunoglobulin. The humanized antibody binds to the same antigen as the donor antibody that provides the CDR. The acceptor framework for humanized immunoglobulins or humanized antibodies can have a limited number of substitutions with amino acids taken from the donor framework. Humanized monoclonal antibodies or other monoclonal antibodies can have additional conserved amino acid substitutions that have virtually no effect on antigen binding or other immunoglobulin functions. Typical conserved substitutions are gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr and other substitutions. Humanized immunoglobulins can be genetically engineered (see, eg, US Pat. No. 5,585,089). Human antibodies are antibodies of which the light chain and heavy chain genes are of human origin. Human antibodies can be produced using methods known in the art. Human antibodies can be produced by immortalizing human B cells that secrete antibodies of interest. Immortalization can be achieved, for example, by infecting EBV or fusing human B cells with myeloma cells or hybridoma cells to generate trioma cells. For human antibodies, see phage presentation methods (eg, Dower et al., PCT Application Publication No. WO 91/17271; McCafferty et al., PCT Application Publication No. WO 92/001047; and Winter, PCT Application Publication No. WO 92/20791; It can also be generated by (see) or by selecting from the Human Combinatrial Monoclonal Antibody Library (see Morphosys website). Human antibodies can also be prepared by using transgenic animals carrying the human immunoglobulin gene (eg, Lonberg et al., PCT Publication No. WO 93/12227; and Kucherlapati, PCT Application Publication No. WO 91/10741. Please refer to).

Thus, the anti-DPP3 antibody or anti-DPP3 antibody fragment according to the invention can have a form known in the art. Examples are, but are not limited to, human antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies, or antibody fragments thereof.

In one particular embodiment of the invention, the anti-DPP3 antibody is a monoclonal antibody or fragment thereof. In one embodiment of the invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment is, or is derived from, a human antibody or humanized antibody. In one particular embodiment, one or more (mouse) CDRs are grafted onto a human antibody or human antibody fragment.

In a preferred embodiment, the antibody of the invention is a recombinantly produced antibody (eg, IgG, which is a typical full-length immunoglobulin) or an antibody containing at least the F variable domain of a heavy chain and / or a light chain. Fragments (eg, chemically bound antibodies (fragment antigen binding); non-limiting examples of chemically bound antibodies include Fab fragments, including Fab minibodies), Single-stranded Fab antibody, monovalent Fab antibody with epitope tag (eg Fab-V5Sx2); divalent Fab (mini-antibody) dimerized with CH ₃ domain; eg multimer with the help of heterologous domains Bivalent or polyvalent Fabs (eg Fab-dHLX-FSx2) formed by (eg, by dimerization of the dHLX domain); F (ab') ₂ fragment, scFv fragment, multimerized Multivalent and / or multispecific scFv fragments, bivalent and / or bispecific deerbodies, BITE® (bispecific T-cell engager), trifunctional antibodies, polyvalent antibodies (eg) Antibodies from a different class than G); single domain antibodies (eg, nanobodies from camel or fish immunoglobulins) and many others.

It is well known that in addition to anti-DPP3 antibodies or anti-DPP3 antibody fragments, other biopolymer scaffolds (so-called non-Ig scaffolds) complex with target molecules and have been used to generate highly target-specific biopolymers. rice field. Examples are aptamers, spiegelmers, anti-quince, and conotoxins.

A protein scaffold is possible as a non-Ig scaffold in the context of the present invention and can be used as an antibody mimetic. This is because non-Ig scaffolds can bind to ligands or antigens. Non-Ig scaffolds are tetranectin-based non-Ig scaffolds (eg, described in United States Patent Application Publication No. 2010/0028995), fibronectin scaffolds (eg, described in EP 1266 025); lipocalin-based scaffolds (described in WO 2011/154420). Ubikitin scaffolds (eg WO 2011/073214), transfer ring scaffolds (eg described in United States Patent Application Publication No. 2004/0023334), protein A scaffolds (eg described in EP 2231860), scaffolds based on ankyrin repeats (eg WO) (Described in 2010/060748), microproteins (preferably microproteins forming cystine knots) scaffolds (eg described in EP 2314308), scaffolds based on the Fyn SH3 domain (eg described in WO 2011/023685), in the EGFR-A domain. You can choose from groups that include scaffolds based on (eg, WO 2005/040229) and scaffolds based on the Knitz domain (eg, EP 1941867). Peptide aptamers or oligonucleotide aptamers are possible as non-Ig scaffolds. Aptamers are usually made by selecting from a large pool of random sequences and are short strands of oligonucleotides (DNA, RNA, or XNA; Xu et al. 2010, Deng et al. 2014) or attached to protein scaffolds. A short variable peptide domain (Li et al., 2011).

In one alternative embodiment, the form of anti-DPP3 antibody is selected from the group comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, F (ab) ₂ fragment, and scFv-Fc fusion protein. In another preferred embodiment, the antibody form is a group comprising scFab fragments, Fab fragments, scFv fragments, and those conjugates with optimized bioavailability (such as PEGylated fragments). Is selected from.

In the context of the present invention, the term "antibody" generally refers to monoclonal antibodies, polyclonal antibodies and their binding fragments (particularly Fc fragments), as well as so-called "single-stranded antibodies" (Bird et al., 1988), chimeric antibodies, humans. Antibodies (especially CDR-grafted antibodies), and dibodies or tetrabodies (Holliger et al., 1993) are included. Also included are immunoglobulin-like proteins selected through techniques including, for example, phage presentation, which specifically bind to the molecule of interest contained in the sample. In this context, the expression "specifically binds" means an antibody that results against a molecule or fragment thereof of interest. Antibodies are considered specific if their affinity for the molecule of interest or fragments thereof is preferably at least 50-fold greater than for other molecules contained in the sample containing the molecule of interest. More preferably 100 times larger, most preferably at least 1000 times larger. In the art, methods for producing antibodies and selecting antibodies with predetermined specificity are well known.

In one particular embodiment of the invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment that binds to the epitope of SEQ ID NO: 2 contained in the DPP3 protein or functional derivative thereof is a monoclonal antibody or a monoclonal antibody fragment thereof. In one embodiment of the invention, the anti-DPP3 antibody or anti-DPP3 antibody fragment that binds to the epitope of SEQ ID NO: 2 contained in the DPP3 protein or functional derivative thereof is derived from a human antibody, or a humanized antibody, or an antibody thereof. An antibody, or a humanized antibody fragment, or a fragment derived from the humanized antibody fragment thereof.

In one particular embodiment, one or more (mouse) CDRs are grafted onto a human antibody or antibody fragment.

In another aspect of the invention, the subject matter provided is an anti-DPP3 antibody or anti-DPP3 antibody fragment thereof grafted with human CDRs towards the epitope of SEQ ID NO: 2 contained in the DPP3 protein or functional derivative thereof. , This anti-DPP3 antibody or anti-DPP3 antibody fragment thereof, which binds to this epitope and is grafted with human CDR, contains an antibody heavy chain variable region (H chain) comprising SEQ ID NO: 4 and / or. It further comprises an antibody light chain variable region (L chain) comprising SEQ ID NO: 5.

Yet another subject of the invention in another aspect is the human CDR-grafted anti-DPP3 antibody or anti-DPP3 antibody fragment thereof towards the epitope of SEQ ID NO: 2 contained in the DPP3 protein or functional derivative thereof. This anti-DPP3 antibody or anti-DPP3 antibody fragment thereof that binds to this epitope and is grafted with human CDR contains an antibody heavy chain variable region (H chain) containing SEQ ID NO: 11 and / or a sequence. It further comprises an antibody light chain variable region (L chain) comprising number 12.

In one particular embodiment of the invention, the subject of the invention is a human monoclonal anti-DPP3 antibody or a monoclonal anti-DPP3 antibody fragment thereof, directed towards the epitope of SEQ ID NO: 2 contained in the DPP3 protein or functional derivative thereof, this epitope. The heavy chain contains at least one CDR of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, and the light chain contains at least 1 of SEQ ID NO: 9, KVS, or SEQ ID NO: 10. Contains two CDRs.

The amount and / or DPP3 activity of the DPP3 protein in the body fluid sample of interest can be determined by a variety of methods (eg, immunoassays, activity assays, mass spectrometry, etc.).

The amount and / or DPP3 activity of the DPP3 protein in the body fluid sample of the subject can be determined, for example, by one of the following methods.

1. 1. Fluorescent Immunoassay (LIA) for quantifying the concentration of DPP3 protein (Rehfeld et al.,
JALM, 2018)

LIA is a single-step chemiluminescent sandwich immunoassay using a high-binding polystyrene microtitration plate as the solid phase. Cover these plates with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). The tracer anti-DPP3 antibody AK2553 is labeled with MA70-acridinium-NHS ester and used at a concentration of 20 ng per well. Pipet a 20 μl sample (eg, serum from the patient's blood, heparin-plasma, citrate-plasma, or EDTA-plasma) and calibrator into a coated white microtitration plate. After adding the tracer antibody AK2553, incubate the microtitration plate at room temperature at 600 rpm for 3 hours. The unbound tracer is then removed by 4 washing steps (350 μl per well). Using a microtitration plate luminescence measuring device, measure the residual chemiluminescence for each well for 1 second. The concentration of DPP3 is determined using a 6-point calibration curve. The calibrator and sample are preferably measured in duplicate.

2. Enzyme Capture Activity Assay (ECA) for Quantifying DPP3 Activity (Rehfeld et al., JALM, 2018)

ECA is a DPP3-specific activity assay using a black high-binding polystyrene microtitration plate as the solid phase. Cover these plates with the monoclonal anti-DPP3 antibody AK2555 (capture antibody). Pipet a 20 μl sample (eg, serum from patient's blood, heparin-plasma, citrate-plasma, EDTA-plasma, cerebrospinal fluid, and urine) and calibrator onto a coated black microdroplet. put in. After adding assay buffer (200 μl), incubate the microtitration plate at 22 ° C. at 600 rpm for 2 hours. Immobilization by binding DPP3 present in the sample to the capture antibody. The unbound components in the sample are removed by 4 washing steps (350 μl per well). After adding the fluorescent substrate Arg-Arg-β-naphthylamide (Arg2-βNA) to the reaction buffer, the specific activity of the immobilized DPP3 is measured by incubating at 37 ° C. for 1 hour. DPP3 specifically cleaves Arg2-βNA to Arg-Arg dipeptide and fluorescent β-naphthylamine. Fluorescence is measured by using a fluorometer at an excitation wavelength of 340 nm and detecting emission at 410 nm. The activity of DPP3 is determined using a 6-point calibration curve. The calibrator and sample are preferably measured in duplicate.

3. 3. Liquid Phase Assay (LAA) for Quantifying DPP3 Activity (Jones et al., Analytical Biochemistry, 1982)

LAA is a liquid phase assay that measures DPP3 activity using a black unbound polystyrene microtitration plate. Pipette 20 μl of sample (eg serum, heparin-plasma, citrate-plasma) and calibrator into a coated black unbound microtitration plate. After adding the fluorescence substrate Arg2-βNA to the assay buffer (200 μl), initial βNA fluorescence (T = 0) is measured by detecting emission at 410 nm using a fluorometer at an excitation wavelength of 340 nm. The plate is then incubated at 37 ° C for 1 hour. The final fluorescence (T = 60) is measured. Calculate the difference between the initial fluorescence and the final fluorescence. The activity of DPP3 is determined using a 6-point calibration curve. The calibrator and sample are preferably measured in duplicate.

The above cutoff values may differ in other assays if they are calibrated differently from the assay system used in the present invention. Therefore, the above cutoff value will be applied in accordance with the assay calibrated in such a different way, taking into account the differences in calibration. One possibility to quantify the difference in calibration is the method-comparative analysis (correlation) of the assay in question and each biomarker assay utilized in the present invention, with each biomarker in the sample used in both methods (eg DPP3). It is carried out by measuring. Another possibility is to utilize the assay in question (assuming this test has sufficient analytical sensitivity) to determine the median biomarker level for each normal population and the results described in the literature. The median biomarker level is compared and the calibration value is recalculated based on the difference obtained by this comparison. When samples from normal (healthy) subjects were measured using the calibration values used in the present invention, the median plasma DPP3 (interquartile range) was 24.6 ng / ml (19.2 ng / ml to 32.4 ng / ml). )Met.

A variety of immunoassays are known and can be utilized in the assays and methods of the invention. Included in the immunoassays are radioimmunoassays (“RIA”), homogenius enzyme-multiplexed immunoassays (“EMIT”), enzyme-bound immunoassay assays (“ELISA”), apoenase reactivation immunoassays (“ARIS”), and chemistry. Luminous and fluorescent immunoassays, Luminex-based bead assays, protein microarray assays, and rapid test formats (eg, radioimmunoassay strip tests (“dipstick immunoassays”), and immunochromatosis assays).

In one embodiment of the invention, such an assay utilizes any type of detection technique, the non-limiting example thereof being an enzyme label, a chemiluminescent label, an electrochemical luminescent label. It is a sandwich immunoassay, preferably a fully automated assay. In one embodiment of the invention, such an assay is an enzyme-labeled sandwich assay. Examples of automated or fully automated assays include the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®: An assay available for one of the Trademarks), Biomerieux Vidas®, and Alere Triage®.

In one embodiment of the invention, a so-called POC (Point of Care) test is possible as the assay. This is a testing technique that allows testing to be performed within an hour near the patient without the need for a fully automated assay system. An example of this technique is an immunochromatography test technique.

In one embodiment of the invention, at least one of the binders is labeled for detection.

In a preferred embodiment, the label is selected from the group comprising a chemiluminescent label, an enzyme label, a fluorescent label, a radioactive iodine label.

Assays can be homogenius or heterogenius, competitive and non-competitive assays. In one embodiment, the assay is a form of sandwich assay that is a non-competitive immunoassay, in which the molecule detected and / or the molecule to be quantified binds to the first and second antibodies. The first antibody can bind to a solid phase (eg, the surface of a bead, well or other container, chip, or strip) and the second antibody is, for example, a dye, a radioisotope, or a reactive or catalytic. It is an antibody labeled with a sexually active moiety. The amount of labeled antibody that binds to the analyte is then measured in an appropriate manner. General configurations and procedures related to the "sandwich assay" have been clarified and are known to those of skill in the art (The Immunoassay Handbook, edited by David Wild, Elsevier LTD, Oxford; 3rd Edition (2005). May), ISBN-13: 978-0080445267; Hultschig C et al., Curr Opin Chem Biol. February 2006; Volume 10 (1): pp. 4-10, PMID: 16376134).

In another embodiment, the assay comprises two capture molecules, preferably antibodies, both of which are present as dispersions in the liquid reaction mixture. In this mixture, the first labeling component is part of a fluorescence-or chemical emission-quenching or amplification based labeling system that attaches to the first trapping molecule and the second labeling of said labeling system. As the component attaches to the second trapped molecule, a measurable signal is generated when both trapped molecules bind to the analyte, thereby detecting the sandwich complex formed in the solution containing the sample. Will be possible.

In another embodiment, the labeling system comprises a rare earth cryptate or rare earth chelate in combination with a fluorescent or chemiluminescent dye, particularly a cyanine-type dye.

In the context of the present invention, fluorescence-based assays include the use of dyes, which include FAM (5-carboxyfluorescein or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-. 700/800, cyanine dyes (CY3, CY5, CY3.5, CY5.5, Cy7, etc.), xanthene, 6-carboxy-2', 4', 7', 4,7-hexachlorofluorescein (HEX), TET, 6-carboxy-4', 5'-dichloro-2', 7'-dimethodifluorescein (JOE), N, N, N', N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy- X-Rhodamine (ROX), 5-Rhodamine-6G (R6G5), 6-Rhodamine-6G (RG6), Rhodamine, Rhodamine Green, Rhodamine Red, Rhodamine 110, BODIPY dyes (BODIPY TMR, etc.), Oregon Green, coumarin (such as Umberiferon), benzimide (such as Hoechst 33258); phenanthridamine (such as Texas Red), Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridinium dye, carbazole dye, phenoxazine dye, porphyrin dye , Polymethine dyes and the like, for example can be selected from the group.

In the context of the present invention, chemiluminescence-based assays include the use of dyes on the basis of physical principles. For the physical principles of chemiluminescent materials, see Kirk-Othmer, Encyclopedia of chemical technology, 4th edition, Editor-in-Chief J.I. Kroschwitz; Editor M. Howe-Grant, John Wiley & Sons, 1993, Vol. 15, It is described on pages 518-562 (incorporated herein by reference, including the citations on pages 551-562). A preferred chemiluminescent dye is an acridinium ester.

As used herein, the "assay" or "diagnostic assay" can be of any type utilized in the field of diagnosis. Such an assay can be based on the binding of the detected analyte to one or more capture probes with a given affinity. With respect to the interaction between the capture molecule and the target or interesting molecule, the affinity constant is preferably greater than 10 ⁸ M ^-1 .

DPP3 activity can be measured by detecting cleavage products of DPP3-specific substrates. Known peptide hormone substrates include Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorfin, proctoline, ACTH (adrenocorticotropic hormone), and MSH (melanocyte stimulating hormone). Hormones; Abramic et al. 2000, Barsun et al. 2007, Dhanda et al. 2008). In addition to the peptide hormone cleavage mentioned, cleavage of other untagged oligopeptides (eg Ala-Ala-Ala-Ala, Dhanda et al. 2008) can be monitored by detecting their respective cleavage products. Non-limiting examples of detection methods include HPLC analysis (eg Lee and Snyder 1982), mass spectrometry (eg Abramic et al. 2000), H ¹ -NMR analysis (eg Vandenberg et al. 1985), capillary zone. Electrophoresis (CE; eg Barsun et al. 2007), thin layer chromatography (eg Dhanda et al. 2008), or reverse phase chromatography (eg Mazocco et al. 2006).

Detection of fluorescence due to hydrolysis of the fluorescent substrate by DPP3 is a standard procedure for monitoring DPP3 activity. These substrates are special dipeptides or tripeptides that couple to fluorophore (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu). -Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe). Non-limiting examples of fluorinated groups include β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide), and 7 -Amid-4-methylcoumarin (AMC, MCA; Abramic et al. 2000, Ohkubo et al. 1999). When these fluorescent substrates are cleaved, fluorescent β-naphthylamide or 7-amide-4-methylcoumarin is released, respectively. In a liquid phase assay or ECA, the substrate and DPP3 are incubated, eg, in the form of a 96-well plate, and fluorescence is measured using a fluorescence detector (Ellis and Nuenke 1967). In addition, a sample containing DPP3 is immobilized on the surface of the gel and divided by electrophoresis, the gel is stained with a fluorescent substrate (eg Arg-Arg-βNA) and Fast Garnet GBC, and the band of fluorescent protein is fluorescent reader. Can be detected by (Ohkubo et al. 1999). Same peptides (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg , Suc-Ala-Ala-Phe) can be coupled to a chromophore (such as p-nitroanilide). DPP3 activity can be monitored by utilizing the detection of color changes due to hydrolysis of the color-developing substrate.

Another option for detecting DPP3 activity is the Protease-Glo ™ assay (commercially available from Promega). In this embodiment of the method described above, a DPP3-specific dipeptide or tripeptide (Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly-Phe, Leu). -Make Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe) to aminoluciferin. When cleaved by DPP3, aminoluciferin is released, acting as a substrate for the bound luciferase to react, releasing detectable luminescence.

In a preferred embodiment, DPP3 activity is measured by adding the fluorescent substrate Arg-Arg-βNA and monitoring the fluorescence in real time.

In a particular embodiment of the method of determining active DPP3 in a body fluid sample of interest, the capture binder that reacts with DPP3 is immobilized on the surface of the solid phase.

When the test sample is passed over a stationary binder, it binds to the binder if DPP3 is present, and DPP3 itself is immobilized and detected. Then, when a substrate is added, the reaction product can be detected, and the reaction product indicates the presence of DPP3 in the test sample or the amount of DPP3. For purposes herein, the term "solid phase" can include any material or container in which the assay can be performed internally or on the surface, and non-limiting examples thereof include porous. Sexual materials, non-porous materials, test tubes, wells, slides, agarose resins (eg Sepharose from GE Healthcare Life Sciences), magnetic particulates (eg Dynabeads ™ or Pierce ™ from Thermo Fisher Scientific) Magnetic beads) and so on.

Binders of protein or protein origin (eg, antibodies, antibody fragments, non-Ig scaffolds) are physically adsorbed (eg, by electrostatic or hydrophobic interactions), bioaffinity immobilization (eg, avidin-biotin, protein A /). Solid phase by a method comprising G / L, His tag, Ni ²⁺ -NTA, GST tag and glutathione, DNA hybridization, aptamer), covalent bond (eg amine and N-hydroxysuccinimide), or a combination of the immobilization methods described above. Immobilize on the surface of. (Kim and Herr 2013). Vinders of oligonucleotide origin (eg, aptamers) can be immobilized on the surface of a solid phase using a (streptavidin) avidin-biotin system (Muller et al. 2012, Deng et al. 2014).

In a particular embodiment of the method of determining the activity of DPP3 in a body fluid sample of interest, the separation step is a washing step of removing components of the sample that are not bound to the capture binder from the captured DPP3. be. As this separation step, any other step of separating DPP3 bound to the capture binder from the components of the body fluid sample is possible.

In a particular embodiment of the method of determining the activity of DPP3 in a body fluid sample of interest, the conversion of the DPP3 substrate by immobilized DPP3 is performed by a fluorescent substrate (eg Arg-Arg-βNA, Arg-Arg-AMC). ) Fluorescence, color change of color-developing substrate, fluorescence of substrate bound to aminoluciferin (Promega's Protease-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography. , Capillary zone electrophoresis, gel electrophoresis followed by active staining (immobilized active DPP3), or measured by a method selected from the group comprising Western blots (cleavage products).

In a particular embodiment of the method for determining the activity of DPP3 in a body fluid sample of interest, the substrates are Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin. , Proctoline, ACTH and MSH, or groups containing dipeptides and tripeptides bound to fluoresceins, chromophores or aminoluciferins (Promega's Protease-Glo ™ assay). Non-limiting examples of dipeptides or tripeptides cleaved by DPP3 include Arg-Arg, Ala-Ala, Ala-Arg, Ala-Phe, Asp-Arg, Gly-Ala, Gly-Arg, Gly. -Phe, Leu-Ala, Leu-Gly, Lys-Ala, Phe-Arg, Suc-Ala-Ala-Phe. Non-limiting examples of fluorinated groups include β-naphthylamide (2-naphthylamide, βNA, 2NA), 4-methoxy-β-naphthylamide (4-methoxy-2-naphthylamide), and 7 -Amid-4-methylcoumarin (AMC, MCA; Abramic et al. 2000, Ohkubo et al. 1999). When these fluorescent substrates are cleaved, fluorescent β-naphthylamide or 7-amide-4-methylcoumarin is released, respectively. A non-limiting example of a chromophore is p-nitroanilide diacetate (pNA). When the peptide-pNA bond in the chromogenic substrate is hydrolyzed, pNA is released, which in turn changes color. Therefore, the change in absorbance (DA / min) is directly proportional to the enzyme activity. Utilizing the Protease-Glo ™ assay from Promega, cleavage by DPP3 releases aminoluciferin, which acts as a substrate for the bound luciferase to react, releasing detectable luminescence.

One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject. Less than:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
It includes a step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.

One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, and methods for determining DPP3 activity in the subject's body fluid sample are as follows:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
It includes a step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.

One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject. The determination comprises the use of a capture binder that specifically binds to full-length DPP3, the capture binder can be selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject. The determination comprises the use of a capture binder that specifically binds to full-length DPP3, the capture binder being an antibody.

One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject. The determination involves the use of a capture binder that specifically binds to full-length DPP3, the capture binder being immobilized on the surface.

One embodiment of the invention is an angiotensin receptor agonist and / or precursor thereof for use in the treatment of disease in a subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined using the method described above. Determined in the body fluid sample of interest, the separation step is a washing step that removes the components of the sample that are not bound to the capture binder from the captured DPP3.

One embodiment of the invention determines the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. The use of methods or assays or kits to do so.

One embodiment of the invention determines the amount and / or DPP3 activity of the DPP3 protein in the body fluid sample of said subject as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. The use of a method or assay or kit for angiotensin receptor agonists and / or precursors thereof, as outlined above, from the group comprising angiotensin I, angiotensin II, angiotensin III, angiotensin IV. Be selected.

One embodiment of the invention is the amount of total DPP3 protein and / or active DPP3 in the body fluid sample of said subject as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. The use of a method or assay or kit to determine the amount of DPP3 protein, wherein the amount and / or the DPP3 activity of the DPP3 protein is prior to and / or during treatment with the angiotensin receptor agonist and / or precursor thereof. In addition, it has been determined at least once in the body fluid sample of the subject.

One embodiment of the invention determines the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. The method or assay or use of a kit for the body fluid sample of subject is selected from whole blood, plasma, and serum.

One embodiment of the invention determines the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject and the following:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein. There is.

One embodiment of the invention determines the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. A method or assay or use of a kit for determining active DPP3 in a body fluid sample of subject.
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding the substrate of DPP3 to the separated DPP3 and then measuring and quantifying the conversion of the substrate of DPP3 is included.
-Includes a step of quantifying the amount of the DPP3 protein.

One embodiment of the invention determines the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. The use of a method or assay or kit to determine the amount and / or DPP3 activity of DPP3 protein in the body fluid sample of the subject, the determination being the use of a capture binder that specifically binds to full-length DPP3. The capture binder can be selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

One embodiment of the invention determines the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. The use of a method or assay or kit to determine the amount and / or DPP3 activity of DPP3 protein in the body fluid sample of the subject, the determination being the use of a capture binder that specifically binds to full-length DPP3. The capture binder is an antibody.

One embodiment of the invention determines the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. The use of a method or assay or kit to determine the amount and / or DPP3 activity of DPP3 protein in the body fluid sample of the subject, the determination being the use of a capture binder that specifically binds to full-length DPP3. The capture binder is immobilized on the surface.

One embodiment of the invention determines the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject and the separation step is the component of the sample not bound to the capture binder. , A cleaning process that removes captured DPP3.

The subject of the present invention is the use of methods, assays or kits for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject, with the aim of predicting the risk of adverse events in said subject. However, the above method is
-Step of determining the level of DPP3 in the body fluid sample of the subject;
-Step of comparing the determined DPP3 level with a predetermined threshold;
• Including the step of correlating the above levels of DPP3 with the risk of adverse events in the subject, elevated levels above a certain threshold predict an increased risk of adverse events.

In the context of the present invention, the term precognition means the prediction of how a patient's condition progresses. It can include speculation of the possibility of recovery or the possibility of an adverse event for the patient.

Adverse events are defined herein as organ dysfunction, or deterioration of organ function, or death.

In the context of the present invention, said subjects are subjects with an increased risk of developing sepsis, subjects suspected of having sepsis, SIRS, sepsis, shock (eg, septic shock), acute and chronic vascular disease (eg, acute). Heart failure, myocardial infarction, stroke, etc.); have a disease selected from the group including organ dysfunction, fluid imbalance, and hypotension of at least one organ.

In a preferred embodiment, the adverse event is organ dysfunction or deterioration of organ function, and the subject is a subject with an increased risk of developing sepsis, a subject suspected of having sepsis, SIRS, sepsis, and shock ( Have a disease selected from the group including (eg, septic shock).

As used herein, organ dysfunction means a condition or health condition in which an organ does not perform its expected function. "Organ dysfunction" means organ dysfunction to the extent that normal homeostasis cannot be maintained without external clinical intervention. The organ failure may be associated with organs selected from the group including kidney, liver, heart, lungs, nervous system. In contrast, organ function represents the expected function of each organ within the physiological range. Those skilled in the art are aware of the function of each organ during a medical examination.

Organ dysfunction can be defined by a set of organ failure assessment scores (SOFA scores) or components thereof. The SOFA score, formerly known as the sepsis-related organ failure assessment score (Singer et al., 2016 JAMA Vol. 315 (8): pp. 801-810), is in the intensive care unit (ICU). It is used to track a person's condition and determine the degree of organ function or the rate of failure. This score is based on six different scores, each related to the respiratory, cardiovascular, hepatic, coagulation, renal, and nervous systems. Each system was given a score of 0-4, with a large score reflecting exacerbation of organ dysfunction. The criteria for assessing the SOFA score are described, for example, by Lambden et al. (See Lambden et al., 2019 Critical Care Vol. 23: 374 for an overview). SOFA scores can traditionally be calculated at the time of admission to the ICU and every 24 hours thereafter.

In particular, the organ dysfunction is selected from the group comprising renal dysfunction, cardiac dysfunction, liver dysfunction, or respiratory tract dysfunction.

In one aspect of the invention, the death occurs after a defined period of time (eg, after 24 hours to 3 months, preferably after 24 hours to 2 months, more preferably after 24 hours to 30 days, Even more preferably after 24 hours to 28 days, even more preferably after 24 hours to 14 days, even more preferably after 24 hours to 7 days, even more preferably after 24 hours to 5 days, even more preferably after 24 hours. It is defined as death after 3 days, most preferably after 24 to 48 hours.

Another particular embodiment of the invention is a method for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject, for the purpose of predicting the risk of adverse events in said subject. Alternatively, the assay or kit is used, and the body fluid sample of the subject is selected from whole blood, plasma, and serum.

One embodiment of the invention is a method or assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject, for the purpose of predicting the risk of adverse events in said subject. The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
It includes a step of quantifying the DPP3 activity by measuring and quantifying the conversion of the DPP3 substrate after adding the DPP3 substrate to the separated DPP3, or a step of quantifying the amount of the DPP3 protein.

Another embodiment of the invention is a method or assay for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. Or the use of a kit and how to determine DPP3 activity in a body fluid sample of interest is
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
It includes a step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.

One embodiment of the invention is a method or assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject, for the purpose of predicting the risk of adverse events in said subject. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, and the determination comprises the use of a capture binder that specifically binds to full-length DPP3. You can choose from a group of antibodies, antibody fragments, or non-IgG scaffolds.

Another embodiment of the invention is a method or assay for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. Or with the use of a kit, the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, said capture binder. Is an antibody.

One embodiment of the invention is a method or assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject, for the purpose of predicting the risk of adverse events in said subject. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is surface. It is fixed to.

Another embodiment of the invention is a method or assay for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. Or with the use of a kit, the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, and the separation step is to remove the components of the sample not bound to the capture binder from the captured DPP3. It is a cleaning step to remove.

Another particular embodiment of the invention is a method for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject, for the purpose of predicting the risk of adverse events in said subject. Alternatively, using an assay or kit, DPP3 activity is determined in the body fluid sample of the subject and the conversion of the DPP3 substrate is the fluorescence, color-developing substrate of a fluorescent substrate (eg, Arg-Arg-βNA, Arg-Arg-AMC). Color change, luminous of substrate bound to aminoluciferin (Promega's Protein-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis , Active staining after gel electrophoresis (immobilized active DPP3), or detected by a method selected from the group comprising Western blots (cleavage products).

Another embodiment of the invention is a method or assay for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. Or with the use of a kit, DPP3 activity is determined in the body fluid sample of the subject and the substrates are angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β- It can be selected from the group comprising casomomorphin, dynorfin, proctrin, ACTH and MSH, or dipeptides bound to phosphors, chromophores or aminoluciferins (where the dipeptide is Arg-Arg).

Another embodiment of the invention is a method or assay for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject, with the aim of predicting the risk of adverse events in said subject. Alternatively, with the use of the kit, DPP3 activity is determined in the body fluid sample of the subject, and the substrate comprises a dipeptide bound to a fluorinated group, chromophore or aminoluciferin (where the dipeptide is Arg-Arg). You can choose from groups.

Another embodiment of the invention is a method for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject, for the purpose of predicting the risk of organ dysfunction or deterioration of organ function. Alternatively, the assay or the use of a kit, the method described above.
-The step of determining the level of DPP3 in the body fluid sample of the subject;
-With the process of comparing the determined DPP3 level with a predetermined threshold;
• Including the step of correlating the above levels of DPP3 with the risk of adverse events in the subject, elevated levels above a certain threshold predict an increased risk of organ dysfunction or deterioration.

Another particular embodiment of the invention is the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. The method or use of an assay or kit to determine, said body fluid sample of subject is selected from whole blood, plasma, and serum.

One embodiment of the invention is for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject, for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. Using a method or assay or kit, the amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein. There is.

Another embodiment of the invention determines the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. A method or assay or use of a kit for determining DPP3 activity in a body fluid sample of said subject.
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
It includes a step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.

Another embodiment of the invention determines the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. The use of a method or assay or kit for which the amount and / or DPP3 activity of DPP3 protein is determined in the body fluid sample of the subject and the determination is the use of a capture binder that specifically binds to full-length DPP3. Including, the capture binder can be selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

Another embodiment of the invention determines the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. The use of a method or assay or kit for which the amount and / or DPP3 activity of DPP3 protein is determined in the body fluid sample of the subject and the determination is the use of a capture binder that specifically binds to full-length DPP3. Including, the capture binder is an antibody.

Another particular embodiment of the invention is the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. The use of a method or assay or kit to determine, where the amount and / or DPP3 activity of DPP3 protein is determined in the body fluid sample of subject, said determination is of a capture binder that specifically binds to full-length DPP3. Including use, the capture binder is immobilized on the surface.

One embodiment of the invention is for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject, for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. Using a method or assay or kit, the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of interest, and the separation step captures the components of the sample that are not bound to the capture binder. This is a cleaning process that removes from DPP3.

Another embodiment of the invention determines the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. For the use of a method or assay or kit for which DPP3 activity is determined in the body fluid sample of the subject and the conversion of the DPP3 substrate is the fluorescence of a fluorescent substrate (eg Arg-Arg-βNA, Arg-Arg-AMC). , Color change of color-developing substrate, Luminescence of substrate bound to aminoluciferin (Promega's Protein-Glo ™ assay), Mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), Thin layer chromatography, Capillary It is detected by a method selected from the group comprising zone electrophoresis, active staining after gel electrophoresis (immobilized active DPP3), or western blots (cleavage products).

Another embodiment of the invention determines the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. DPP3 activity is determined in the body fluid sample of the subject and the substrates are angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2. , Valolfin, β-casomorphin, dynorfin, proctoline, ACTH and MSH, or a group comprising dipeptides bound to fluorinated groups, chromophores or aminoluciferins (where the dipeptide is Arg-Arg). ..

One embodiment of the present invention is for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of the subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in the subject. The use of a method or assay or kit, where DPP3 activity is determined in the body fluid sample of the subject and the substrate is a dipeptide bound to a phosphor, chromophore or aminoluciferin (where the dipeptide is Arg-Arg). Can be selected from groups that include.

The subject of the present invention is also a method of predicting the risk of an adverse event in the subject, wherein the method is:
-Step of determining the level of DPP3 in the body fluid sample of the subject;
-Step of comparing the determined DPP3 level with a predetermined threshold;
• Including the step of correlating the above levels of DPP3 with the risk of adverse events in the subject, elevated levels above a certain threshold predict an increased risk of adverse events.

The subject of the present invention is also a method of predicting the risk of adverse events in the subject.

Another embodiment of the invention is a method of predicting the risk of adverse events in the subject, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum.

Another particular embodiment of the invention is a method of predicting the risk of adverse events in said subject, where the amount and / or DPP3 activity of DPP3 protein is determined in the subject's body fluid sample, and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein. There is.

Another embodiment of the invention is a method of predicting the risk of adverse events in the subject and a method of determining DPP3 activity in the subject's body fluid sample.
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
It includes a step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.

Another embodiment of the invention is a method of predicting the risk of adverse events in said subject, where the amount and / or DPP3 activity of DPP3 protein is determined in the subject's body fluid sample, the determination being full length. It comprises the use of a capture binder that specifically binds to DPP3, said capture binder can be selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

Another embodiment of the invention is a method of predicting the risk of adverse events in said subject, where the amount and / or DPP3 activity of DPP3 protein is determined in the subject's body fluid sample, the determination being full length. The capture binder comprises the use of a capture binder that specifically binds to DPP3, said capture binder being an antibody.

Another embodiment of the invention is a method of predicting the risk of adverse events in said subject, where the amount and / or DPP3 activity of DPP3 protein is determined in the subject's body fluid sample, the determination being full length. The capture binder comprises the use of a capture binder that specifically binds to DPP3, the capture binder being immobilized on the surface.

Another embodiment of the invention is a method of predicting the risk of adverse events in the subject, where the amount and / or DPP3 activity of the DPP3 protein is determined in the subject's body fluid sample and the separation step is the subject. This is a cleaning step that removes the components of the sample that are not bound to the capture binder from the captured DPP3.

Another embodiment of the invention is a method of predicting the risk of adverse events in the subject, where DPP3 activity is determined in the subject's body fluid sample and conversion of the DPP3 substrate is performed by a fluorescent substrate (eg, Arg-). Fluorescence of Arg-βNA, Arg-Arg-AMC), color change of color-developing substrate, luminescence of substrate bound to aminoluciferin (Promega's Protease-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase) By a method selected from the group comprising chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, active staining after gel electrophoresis (immobilized active DPP3), or western blot (cut product). Detected.

Yet another embodiment of the invention is a method of predicting the risk of adverse events in the subject, where DPP3 activity is determined in the subject's body fluid sample and the substrates are angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or dipeptides bound to phosphors, chromophores or aminoluciferins (although the dipeptide is Arg- You can choose from groups that include (Arg).

One embodiment of the invention is a method of predicting the risk of adverse events in the subject, where DPP3 activity is determined in the subject's body fluid sample and the substrate is a dipeptide bound to fluorescein, chromophore or aminoluciferin. (However, the dipeptide is Arg-Arg) can be selected from the group containing.

One embodiment of the present invention is a method for predicting the risk of organ dysfunction or deterioration of organ function in the subject.
-Step of determining the level of DPP3 in the body fluid sample of the subject;
-Step of comparing the determined DPP3 level with a predetermined threshold;
• Including the step of correlating the above levels of DPP3 with the risk of adverse events in the subject, elevated levels above a certain threshold predict an increased risk of organ dysfunction or deterioration.

Another particular embodiment of the invention is a method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum. ..

One embodiment of the invention is a method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein. There is.

Another embodiment of the present invention is a method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, and a method of determining DPP3 activity in the body fluid sample of the subject.
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
It includes a step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.

Another particular embodiment of the invention is a method of predicting the risk of organ dysfunction or deterioration of organ function in said subject, where the amount and / or DPP3 activity of DPP3 protein is determined in the subject's body fluid sample. The determination comprises the use of a capture binder that specifically binds to full-length DPP3, the capture binder can be selected from the group of antibodies, antibody fragments, or non-IgG scaffolds.

Yet another embodiment of the invention is a method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject. The determination comprises the use of a capture binder that specifically binds to full-length DPP3, the capture binder being an antibody.

One embodiment of the invention is a method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, said determination. The capture binder is immobilized on the surface, including the use of a capture binder that specifically binds to full-length DPP3.

Another embodiment of the invention is a method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject. The separation step is a cleaning step of removing the components of the sample that are not bound to the capture binder from the captured DPP3.

Another particular embodiment of the invention is a method of predicting the risk of organ dysfunction or deterioration of organ function in said subject, where DPP3 activity is determined in the subject's body fluid sample and conversion of the substrate of DPP3 is performed. , Fluorescence of fluorescent substrates (eg Arg-Arg-βNA, Arg-Arg-AMC), color change of color-developing substrate, luminescence of substrate bound to aminoluciferin (Promega Protease-Glo ™ assay), mass analysis. , HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, active staining after gel electrophoresis (immobilized active DPP3), or western blot (cut product). Detected by the method selected from the group.

Another particular embodiment of the invention is a method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, where DPP3 activity is determined in the subject's body fluid sample and the substrate is angiotensin II. , III, and IV, Leu-enkephalin, Met-enkephalin, endomorphin 1 and 2, barolphin, β-casomorphin, dynorphin, proctrin, ACTH and MSH, or dipeptides bound to phosphors, chromophores or aminoluciferins (although , The dipeptide is Arg-Arg).

Another embodiment of the present invention is a method for predicting the risk of organ dysfunction or deterioration of organ function in the subject, DPP3 activity is determined in the body fluid sample of the subject, and the substrate is a chromophore, chromophore. It can be selected from a group or a group containing a dipeptide bound to aminoluciferin (where the dipeptide is Arg-Arg).

Yet another embodiment of the present invention is as follows.

The use of angiotensin receptor agonists and / or precursors thereof in the manufacture of drugs to treat disease in a subject.
-The diseases include heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial disease, viral disease (eg AIDS), parasite disease (eg AIDS)). Selected from groups including malaria))), SIRS or septicemia, cancer, acute renal failure (AKI), CNS disorders (eg, seizures, neurodegenerative disorders), autoimmune disorders, vascular disorders, hypotension, and shock.
The subject has an amount and / or DPP3 activity of DPP3 protein in a body fluid sample that exceeds a predetermined threshold, use.

Item 1. The use according to Item 1, wherein the angiotensin receptor agonist and / or its precursor is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, and angiotensin IV, in particular angiotensin II.

The amount and / or the DPP3 activity of the DPP3 protein has been determined at least once in the body fluid sample of the subject before and / or during treatment with the angiotensin receptor agonist and / or precursor thereof. Use as described in Section 1 or 2.

The determination of the amount and / or DPP3 activity of the DPP3 protein determined in the body fluid sample of the subject is used as a therapeutic guide and / or therapeutic monitoring of the treatment with angiotensin receptor agonists and / or precursors thereof, Item 1. Use described in any one of 3 to 3.

Item 6. The use according to any one of Items 1 to 4, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum.

Item 6. The use according to any one of Items 1 to 5, wherein the angiotensin receptor agonist and / or a precursor thereof is administered to the subject as a pharmaceutical preparation.

Item 6. The use according to Item 6, wherein the angiotensin receptor agonist and / or its precursor is angiotensin II acetate, and the pharmaceutical product is in a dry state where it is reconstituted before use.

The angiotensin receptor agonist and / or its precursor is 0.1 to 200 ng / kg / min, preferably 1 to 100 ng / kg / min, more preferably 2 to 80 ng / kg / min, still more preferably 5 to Administered at a rate of 60 ng / kg / min, even more preferably 10-50 ng / kg / min, even more preferably 15-40 ng / kg / min, most preferably 20 ng / kg / min. The use according to any one of items 1 to 7, which is angiotensin II.

Item 6. The use according to any one of Items 1 to 8, wherein the angiotensin receptor agonist and / or its precursor, particularly angiotensin II, is administered in combination with an inhibitor of DPP3.

Item 9. The use according to Item 9, wherein the inhibitor of DPP3 is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

Item 9. The use according to Item 9 or 10, wherein the inhibitor of DPP3 is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 1, in particular SEQ ID NO: 2.

The use according to any one of Items 9 to 11, wherein the inhibitor of DPP3 is an antibody or fragment or scaffold that exhibits a minimum binding affinity of ^10-7 M or less for DPP3.

Item 6. The use according to any one of Items 9 to 12, wherein the inhibitor of DPP3 is a unispecific antibody or fragment or scaffold.

The inhibitor of DPP3 binds to full-length DPP3 and increases the activity of DPP3 by at least 10%, or at least 50%, more preferably at least 60%, even more preferably greater than 70%, even more preferably greater than 80%, further. Item 6. The use according to any one of Items 9 to 13, which is an antibody or fragment or scaffold that suppresses more than 90%, more preferably more than 95%.

The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein. Use according to any one of Items 1 to 14.

The method of determining DPP3 activity in the body fluid sample of the subject is:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
The use according to any one of Items 1 to 15, comprising the step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.

The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, antibody fragment,. Or the use according to any one of Items 1-16, selected from the group of non-IgG scaffolds.

The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, item 1. Use described in any one of 17 to 17.

The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is immobilized on the surface. Yes, the use described in any one of items 1 to 18.

The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of interest, and the separation step is a washing step of removing components of the sample that are not bound to the capture binder from the captured DPP3. , The use according to any one of Items 16 to 19.

DPP3 activity was determined in the body fluid sample of interest, and conversion of the DPP3 substrate was associated with fluorescence of the fluorescent substrate (eg Arg-Arg-βNA, Arg-Arg-AMC), color change of the color-developing substrate, aminoluciferin. Active staining after substrate fluorescence (Promega Protease-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis 28. The use according to any one of Items 16-20, which is detected by a method selected from the group comprising (immobilized active DPP3) or Western blots (cleavage products).

DPP3 activity is determined in the body fluid sample of the subject, and the substrates are angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin, proctrin, Item 6. The use according to any one of Items 16 to 21, selected from the group comprising ACTH and MSH, or a dipeptide bound to a fluorescent group, chromophore or enkephalin (where the dipeptide is Arg-Arg). ..

The DPP3 activity is determined in the body fluid sample of interest and the substrate is selected from the group comprising a dipeptide bound to a fluorophore, chromophore or aminoluciferin (where the dipeptide is Arg-Arg). Use as described in any one of 16 to 22.

A method of treating a disease in a subject, comprising administering to the subject an angiotensin receptor agonist and / or a precursor thereof.
-The diseases include heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial disease, viral disease (eg AIDS), parasite disease (eg AIDS)). Selected from groups including malaria))), SIRS or septicemia, cancer, acute renal failure (AKI), CNS disorders (eg, seizures, neurodegenerative disorders), autoimmune disorders, vascular disorders, hypotension, and shock.
A method in which the subject has an amount and / or DPP3 activity of DPP3 protein in a body fluid sample that exceeds a predetermined threshold.

Item 5. The method of item 1, wherein the angiotensin receptor agonist and / or precursor thereof is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, and angiotensin IV, in particular angiotensin II.

The amount and / or the DPP3 activity of the DPP3 protein is determined at least once in the body fluid sample of the subject before and / or during treatment with the angiotensin receptor agonist and / or precursor thereof. Yes, the method according to item 1 or 2.

The determination of the amount and / or DPP3 activity of DPP3 protein determined in the body fluid sample of the subject is used as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, Item 1-. The method described in any one of 3.

Item 6. The method according to any one of Items 1 to 4, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum.

Item 6. The method according to any one of Items 1 to 5, wherein the angiotensin receptor agonist and / or a precursor thereof is administered to the subject as a pharmaceutical preparation.

Item 6. The method according to Item 6, wherein the angiotensin receptor agonist and / or its precursor is angiotensin II acetate, and the pharmaceutical product is in a dry state in which it is reconstituted before use.

The angiotensin receptor agonist and / or its precursor is 0.1 to 200 ng / kg / min, preferably 1 to 100 ng / kg / min, more preferably 2 to 80 ng / kg / min, still more preferably 5 to Administered at a rate of 60 ng / kg / min, even more preferably 10-50 ng / kg / min, even more preferably 15-40 ng / kg / min, most preferably 20 ng / kg / min. The method according to any one of Items 1 to 7, which is angiotensin II.

Item 6. The method according to any one of Items 1 to 8, wherein the angiotensin receptor agonist and / or a precursor thereof, particularly angiotensin II, is administered in combination with an inhibitor of DPP3.

Item 9. The method of Item 9, wherein the inhibitor of DPP3 is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold.

Item 9. The method of item 9 or 10, wherein the inhibitor of DPP3 is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 1, in particular SEQ ID NO: 2.

Item 6. The method according to any one of Items 9 to 11, wherein the inhibitor of DPP3 is an antibody or fragment or scaffold that exhibits a minimum binding affinity of ^10-7 M or less for DPP3.

Item 6. The method according to any one of Items 9 to 12, wherein the inhibitor of DPP3 is a unispecific antibody or fragment or scaffold.

The inhibitor of DPP3 binds to full-length DPP3 and increases the activity of DPP3 by at least 10%, or at least 50%, more preferably at least 60%, even more preferably greater than 70%, even more preferably greater than 80%, further. Item 6. The method according to any one of Items 9 to 13, wherein the antibody or fragment or scaffold is more preferably more than 90%, still more preferably more than 95% inhibitory.

The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein. The method according to any one of Items 1 to 14.

The method of determining DPP3 activity in the body fluid sample of the subject is:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
Item 6. The method according to any one of Items 1 to 15, comprising a step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.

The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, antibody fragment,. The method according to any one of Items 1 to 16, which is selected from the group of non-IgG scaffolds.

The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, item 1. The method according to any one of 17 to 17.

The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is immobilized on the surface. The method according to any one of Items 1 to 18.

The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of interest, and the separation step is a washing step of removing components of the sample that are not bound to the capture binder from the captured DPP3. The method according to any one of Items 16 to 19.

DPP3 activity was determined in the body fluid sample of the subject, and conversion of the DPP3 substrate was associated with fluorescence of the fluorescent substrate (eg Arg-Arg-βNA, Arg-Arg-AMC), color change of the color-developing substrate, aminoluciferin. Active staining after substrate fluorescence (Promega Protease-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis Item 6. The method according to any one of Items 16 to 20, which is detected by a method selected from a group comprising (immobilized active DPP3) or a western blot (cleavage product).

DPP3 activity is determined in the body fluid sample of the subject, and the substrates are angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin, proctrin, Item 6. The method according to any one of Items 16 to 21, selected from the group comprising ACTH and MSH, or a dipeptide bound to a fluorescent group, chromophore or enkephalin (where the dipeptide is Arg-Arg). ..

The DPP3 activity is determined in the body fluid sample of interest and the substrate is selected from the group comprising a dipeptide bound to a fluorophore, chromophore or aminoluciferin (where the dipeptide is Arg-Arg). The method according to any one of 16 to 22.

Yet another embodiment of the present invention is as follows.

1. 1. Angiotensin receptor agonist and / or precursor thereof for use in the treatment of disease in a subject.
-The diseases include heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial disease, viral disease (eg AIDS), parasite disease (eg AIDS)). Selected from groups including malaria))), SIRS or septicemia, cancer, acute renal failure (AKI), CNS disorders (eg, seizures, neurodegenerative disorders), autoimmune disorders, vascular disorders, hypotension, and shock.
The subject is an angiotensin receptor agonist and / or precursor thereof having an amount and / or DPP3 activity of DPP3 protein in a body fluid sample that exceeds a predetermined threshold.

2. Item 2. Angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in a subject selected from the group comprising angiotensin I, angiotensin II, angiotensin III, and angiotensin IV, in particular angiotensin II. body.

3. 3. The amount and / or the DPP3 activity of the DPP3 protein has been determined at least once in the body fluid sample of the subject before and / or during treatment with the angiotensin receptor agonist and / or precursor thereof. Angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in the subject according to Item 1 or 2.

Four. The determination of the amount and / or DPP3 activity of the DPP3 protein determined in the body fluid sample of the subject is used as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of 3.

Five. Angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in the subject according to any one of Items 1 to 4, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum. body.

6. Item 5. The angiotensin receptor agonist and / or the angiotensin receptor agonist according to any one of Items 1 to 5, wherein the angiotensin receptor agonist and / or a precursor thereof is administered to the subject in a pharmaceutical preparation and is used for treating a disease in the subject. Or its precursor.

7. 7. Item 6. Angiotensin for use in the treatment of a disease in the subject according to Item 6, wherein the angiotensin receptor agonist and / or its precursor is angiotensin II acetate and the pharmaceutical preparation is in a dry state reconstituted before use. Receptor agonists and / or precursors thereof.

8.0.1 to 200 ng / kg / min, preferably 1 to 100 ng / kg / min, more preferably 2 to 80 ng / kg / min, even more preferably 5 to 60 ng / kg / min, even more preferred. Is angiotensin II administered at a rate of 10-50 ng / kg / min, even more preferably 15-40 ng / kg / min, most preferably 20 ng / kg / min, items 1-7. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of the above.

9. Angiotensin for use in the subject of any one of Items 1-8, wherein the angiotensin receptor agonist and / or precursor thereof, in particular angiotensin II, is administered in combination with an inhibitor of DPP3. Receptor agonists and / or precursors thereof.

Ten. The angiotensin receptor agonist and / or an angiotensin receptor agonist for use in the treatment of a disease in the subject according to Item 9, wherein the inhibitor of DPP3 is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold. Its precursor.

11. 11. Item 9. Treatment of a disease in a subject according to Item 9 or 10, wherein the inhibitor of DPP3 is either an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 1, in particular SEQ ID NO: 2. Angiotensin receptor agonists and / or precursors thereof for use in.

12. Used in the treatment of a disease in a subject according to any one of Items 9 to 11, wherein the inhibitor of DPP3 is an antibody or fragment or scaffold that exhibits a minimum binding affinity of ^10-7 M or less for DPP3. Angiotensin receptor agonist and / or precursor thereof.

13. Angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in the subject according to any one of Items 9 to 12, wherein the inhibitor of DPP3 is a unispecific antibody or fragment or scaffold. body.

14. The inhibitor of DPP3 binds to full-length DPP3 and increases the activity of DPP3 by at least 10%, or at least 50%, more preferably at least 60%, even more preferably greater than 70%, even more preferably greater than 80%. Angiotensin receptor agonist for use in the treatment of a disease in the subject of any one of Items 9-13, which is an antibody or fragment or scaffold that suppresses even more preferably> 90%, even more preferably> 95%. And / or its precursor.

15. 15. The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein.
Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 1 to 14.

16. The method of determining DPP3 activity in the body fluid sample of the subject is:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
Including the step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.
Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 1 to 15.

17. 17. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, antibody fragment,. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 1 to 16, selected from the group of non-IgG scaffolds.

18. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, item 1. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of 17 to 17.

19. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, and the determination comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is immobilized on the surface. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 1 to 18.

20. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of interest, and the separation step is a washing step of removing components of the sample that are not bound to the capture binder from the captured DPP3. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 16 to 19.

twenty one. DPP3 activity was determined in the body fluid sample of the subject, and conversion of the DPP3 substrate was associated with fluorescence of the fluorescent substrate (eg Arg-Arg-βNA, Arg-Arg-AMC), color change of the color-developing substrate, and aminoluciferin. Active staining after substrate luminescence (Promega Protease-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis (Immobilized active DPP3), or for use in the treatment of the disease in the subject according to any one of Items 16-20, detected by a method selected from the group comprising Western blots (cleavage products). Angiotensin receptor agonist and / or a precursor thereof.

twenty two. DPP3 activity is determined in the body fluid sample of the subject and the substrates are angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin, proctoline, Item 5. The subject according to any one of Items 16 to 21, selected from the group comprising ACTH and MSH, or a dipeptide bound to a phosphor, chromophore or enkephalin (where the dipeptide is Arg-Arg). Angiotensin receptor agonists and / or precursors thereof for use in the treatment of diseases in.

twenty three. The DPP3 activity is determined in the body fluid sample of interest and the substrate is selected from the group comprising dipeptides bound to fluoresceins, chromophores or aminoluciferins (where the dipeptide is Arg-Arg). Angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in the subject according to any one of 16 to 22.

twenty four. A method or assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. Use of.

twenty five. The angiotensin receptor agonist and / or its precursor is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, and angiotensin IV, particularly angiotensin II, the angiotensin receptor agonist and / or its precursor. Use of the method or assay or kit according to Section 24 for determining the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with.

26. The amount and / or DPP3 activity of the DPP3 protein is determined at least once in the body fluid sample of the subject before and / or during treatment with the angiotensin receptor agonist and / or precursor thereof. 28. The method or assay or kit of Section 24 or 25 for determining the amount and / or DPP3 activity of the DPP3 protein as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. Use of.

27. The amount and / of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum. Alternatively, use of the method, assay or kit according to any one of Sections 24-26 for determining DPP3 activity.

28. The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein.
28. Item 1. Use of methods or assays or kits.

29. The method of determining DPP3 activity in the body fluid sample of the subject is:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-The process of separating DPP3 bound to the capture binder and
-The step of adding the substrate of DPP3 to the separated DPP3 and
Including the step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.
21. Item 25-28 for determining the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. Use of methods or assays or kits.

30. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, antibody fragment,. Or a section for determining the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, selected from the group of non-IgG scaffolds. Use of the method or assay or kit according to 28 or 29.

31. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, and the determination comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, angiotensin receptor. The method according to any one of Sections 28-30 for determining the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with a body agonist and / or precursor thereof. Use of an assay or kit.

32. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is immobilized on the surface. In any one of Sections 28-31 for determining the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. Use of the described method or assay or kit.

33. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of interest, and the separation step is a washing step of removing components of the sample that are not bound to the capture binder from the captured DPP3. 28-32 of item 1 for determining the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. Use of methods or assays or kits.

34. DPP3 activity was determined in the body fluid sample of interest, and conversion of the DPP3 substrate was associated with fluorescence of the fluorescent substrate (eg Arg-Arg-βNA, Arg-Arg-AMC), color change of the color-developing substrate, and aminoluciferin. Active staining after substrate luminescence (Promega's Protein-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis A treatment guide and / or treatment guide for treatment with angiotensin receptor agonists and / or precursors thereof, detected by a method selected from the group comprising (immobilized active DPP3), or western blots (cleavage products). Use of the method or assay or kit according to any one of Sections 28-33 for determining the amount and / or DPP3 activity of DPP3 protein as therapeutic monitoring.

35. DPP3 activity is determined in the body fluid sample of the subject and the substrates are angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin, protein, Treatment with angiotensin receptor agonists and / or precursors selected from the group comprising ACTH and MSH, or dipeptides bound to fluorescent groups, chromophores or aminoluciferins (where the dipeptide is Arg-Arg). Use of the method or assay or kit according to any one of Sections 28-34 for determining the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for.

36. Angiotensin, whose DPP3 activity is determined in the body fluid sample of interest, is selected from the group in which the substrate comprises a dipeptide bound to a fluorescent group, chromophore or aminoluciferin (where the dipeptide is Arg-Arg). Item 6. The method according to any one of Items 28-35 for determining the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with a receptor agonist and / or a precursor thereof. Or use of an assay or kit.

37. The treatment with angiotensin receptor agonists and / or precursors thereof is discontinued when the patient has an amount and / or DPP3 activity of DPP3 protein in a body fluid sample below a predetermined threshold level. , Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 1 to 23.

38. When the subject has an amount and / or DPP3 activity of DPP3 protein in a body fluid sample above a predetermined threshold level, the treatment with angiotensin receptor agonist and / or precursor thereof is initiated, Item 1-. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of 23 and 27.

In the above context, the following serially numbered embodiments provide further specific aspects of the invention.

1. 1. Angiotensin receptor agonist and / or precursor thereof for use in the treatment of disease in a subject.
-The diseases include heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial disease, viral disease (eg AIDS), parasite disease (eg AIDS)). Selected from groups including malaria))), SIRS or septicemia, cancer, acute renal failure (AKI), CNS disorders (eg, seizures, neurodegenerative disorders), autoimmune disorders, vascular disorders, hypotension, and shock.
The subject is an angiotensin receptor agonist and / or precursor thereof having an amount and / or DPP3 activity of DPP3 protein in a body fluid sample that exceeds a predetermined threshold.

2. Angiotensin receptor agonists and / or precursors thereof for use in the treatment of diseases in the subject according to Item 1, selected from the group comprising angiotensin I, angiotensin II, angiotensin III, and angiotensin IV, in particular angiotensin II. body.

3. 3. The amount and / or the DPP3 activity of the DPP3 protein has been determined at least once in the body fluid sample of the subject before and / or during treatment with the angiotensin receptor agonist and / or precursor thereof. Angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in the subject according to Item 1 or 2.

Four. The determination of the amount and / or DPP3 activity of DPP3 protein determined in the body fluid sample of the subject is used as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of 1 to 3.

Five. Angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in the subject according to any one of Items 1 to 4, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum. body.

6. Angiotensin receptor agonist and / or angiotensin receptor agonist for use in the treatment of a disease in the subject according to any one of Items 1 to 5, wherein the angiotensin receptor agonist and / or a precursor thereof is administered to the subject in a pharmaceutical preparation. Or its precursor.

7. 7. Item 6. Angiotensin for use in the treatment of a disease in the subject according to Item 6, wherein the angiotensin receptor agonist and / or its precursor is angiotensin II acetate and the pharmaceutical preparation is in a dry state reconstituted before use. Receptor agonists and / or precursors thereof.

8. The angiotensin receptor agonist and / or precursor thereof is 0.1 to 200 ng / kg / min, preferably 1 to 100 ng / kg / min, more preferably 2 to 80 ng / kg / min, and even more preferably 5 Administered at a rate of ~ 60 ng / kg / min, even more preferably 10-50 ng / kg / min, even more preferably 15-40 ng / kg / min, most preferably 20 ng / kg / min. Angiotensin II, an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of items 1 to 7.

9. Angiotensin for use in the subject of any one of Items 1-8, wherein the angiotensin receptor agonist and / or precursor thereof, in particular angiotensin II, is administered in combination with an inhibitor of DPP3. Receptor agonists and / or precursors thereof.

Ten. The angiotensin receptor agonist and / or an angiotensin receptor agonist for use in the treatment of a disease in the subject according to Item 9, wherein the inhibitor of DPP3 is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold. Its precursor.

11. 11. To be used in the treatment of a disease in a subject according to Item 9 or 10, wherein the inhibitor of DPP3 is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 1, in particular SEQ ID NO: 2. Angiotensin receptor agonist and / or its precursor.

12. Used in the treatment of a disease in a subject according to any one of Items 9 to 11, wherein the inhibitor of DPP3 is an antibody or fragment or scaffold that exhibits a minimum binding affinity of ^10-7 M or less for DPP3. Angiotensin receptor agonist and / or precursor thereof.

13. Angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in the subject according to any one of Items 9 to 12, wherein the inhibitor of DPP3 is a unispecific antibody or fragment or scaffold. body.

14. The inhibitor of DPP3 binds to full-length DPP3 and increases the activity of DPP3 by at least 10%, or at least 50%, more preferably at least 60%, even more preferably greater than 70%, even more preferably greater than 80%. Angiotensin receptor agonist for use in the treatment of a disease in the subject according to any one of Items 9 to 13, which is an antibody or fragment or scaffold that suppresses more than 90%, even more preferably more than 95%. And / or its precursor.

15. 15. The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein.
Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 1 to 14.

16. The method of determining DPP3 activity in the body fluid sample of the subject is:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
Including the step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.
Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 1 to 15.

17. 17. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, antibody fragment,. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 1 to 16, selected from the group of non-IgG scaffolds.

18. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, item 1. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in a subject according to any one of 17 to 17.

19. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, and the determination comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is immobilized on the surface. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 1 to 18.

20. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of interest, and the separation step is a washing step of removing components of the sample that are not bound to the capture binder from the captured DPP3. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 16 to 19.

twenty one. DPP3 activity was determined in the body fluid sample of the subject, and conversion of the DPP3 substrate was associated with fluorescence of the fluorescent substrate (eg Arg-Arg-βNA, Arg-Arg-AMC), color change of the color-developing substrate, and aminoluciferin. Active staining after substrate luminescence (Promega Protease-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis (Immobilized active DPP3), or for use in the treatment of the disease in the subject according to any one of Items 16-20, detected by a method selected from the group comprising Western blots (cleavage products). Angiotensin receptor agonist and / or a precursor thereof.

twenty two. DPP3 activity is determined in the body fluid sample of the subject and the substrates are angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin, proctoline, Item 5. The subject according to any one of Items 16 to 21, selected from the group comprising ACTH and MSH, or a dipeptide bound to a phosphor, chromophore or enkephalin (where the dipeptide is Arg-Arg). Angiotensin receptor agonists and / or precursors thereof for use in the treatment of diseases in.

twenty three. The DPP3 activity is determined in the body fluid sample of interest and the substrate is selected from the group comprising dipeptides bound to fluoresceins, chromophores or aminoluciferins (where the dipeptide is Arg-Arg). Angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in the subject according to any one of 16 to 22.

twenty four. A method or assay for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. Use of the kit.

twenty five. Item 24. The angiotensin receptor agonist according to Item 24, wherein the angiotensin receptor agonist and / or its precursor is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, and angiotensin IV, particularly angiotensin II. Use of methods or assays or kits for determining the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with and / or its precursors.

26. The amount and / or DPP3 activity of the DPP3 protein is determined at least once in the body fluid sample of the subject before and / or during treatment with the angiotensin receptor agonist and / or precursor thereof. A method or assay for determining the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, as described in Section 24 or 25. Or use the kit.

27. The treatment guide for treatment with angiotensin receptor agonist and / or precursor thereof according to any one of Items 24 to 26, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum. And / or use of methods or assays or kits for determining the amount and / or DPP3 activity of DPP3 protein as therapeutic monitoring.

28. The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein.
Determine the amount and / or DPP3 activity of the DPP3 protein as a treatment guide and / or treatment monitoring for treatment with the angiotensin receptor agonist and / or precursor thereof according to any one of Items 24-27. For the use of methods or assays or kits.

29. The method of determining DPP3 activity in the body fluid sample of the subject is:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
Including the step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.
Determine the amount and / or DPP3 activity of the DPP3 protein as a treatment guide and / or treatment monitoring for treatment with the angiotensin receptor agonist and / or precursor thereof according to any one of Items 24-28. For the use of methods or assays or kits.

30. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, antibody fragment,. Or the amount of DPP3 protein and / or as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, according to item 28 or 29, selected from the group of non-IgG scaffolds. Or the use of methods or assays or kits to determine DPP3 activity.

31. Item 28, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody. A method for determining the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with the angiotensin receptor agonist and / or precursor thereof according to any one of 30 to 30. Or use an assay or kit.

32. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is immobilized on the surface. The amount and / or DPP3 activity of the DPP3 protein as a treatment guide and / or treatment monitoring for treatment with the angiotensin receptor agonist and / or precursor thereof according to any one of Items 28 to 31. Use of methods or assays or kits to determine.

33. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of interest, and the separation step is a washing step of removing components of the sample that are not bound to the capture binder from the captured DPP3. Determine the amount and / or DPP3 activity of the DPP3 protein as a treatment guide and / or treatment monitoring for treatment with the angiotensin receptor agonist and / or precursor thereof according to any one of Items 28-32. For the use of methods or assays or kits.

34. DPP3 activity was determined in the body fluid sample of interest, and conversion of the DPP3 substrate was associated with fluorescence of the fluorescent substrate (eg Arg-Arg-βNA, Arg-Arg-AMC), color change of the color-developing substrate, and aminoluciferin. Active staining after substrate luminescence (Promega's Protein-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis Item 5. Angiotensin receptor agonist according to any one of Items 28 to 33, which is detected by a method selected from the group containing (immobilized active DPP3) or a western blot (cleavage product) and / or its own. Use of methods or assays or kits for determining the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with precursors.

35. DPP3 activity is determined in the body fluid sample of the subject and the substrates are angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin, protein, Item 12. Angiotensin according to any one of Items 28 to 34, which is selected from the group containing ACTH and MSH, or a dipeptide bound to a fluorescent group, a chromogen or an aminoluciferin (where the dipeptide is Arg-Arg). Use of methods or assays or kits for determining the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with receptor agonists and / or precursors thereof.

36. The DPP3 activity is determined in the body fluid sample of interest and the substrate is selected from the group comprising a dipeptide bound to a fluorescent group, chromophore or angiotensin (where the dipeptide is Arg-Arg). To determine the amount and / or DPP3 activity of DPP3 protein as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, as described in any one of 28-35. Method or use of assay or kit.

37. The use of a method, assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. but,
-Step of determining the level of DPP3 in the body fluid sample of the subject;
-Step of comparing the determined DPP3 level with a predetermined threshold;
The step of correlating the level of DPP3 with the risk of adverse events in the subject,
Use, where elevated levels above a certain threshold predict an increased risk of said adverse event, including.

38. Use of a method, assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. The body fluid sample of is selected from whole blood, plasma, and serum, used.

39. Use of a method, assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. The amount and / or DPP3 activity of the subject was determined in the body fluid sample of the subject, and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein. use.

40. Use of a method, assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. Is determined in the body fluid sample of the subject.
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
Use, including the step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.

41. Use of a method, assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. And / or DPP3 activity is determined in the body fluid sample of the subject, said determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, antibody fragment, or non-antibody. Used, selected from the group of IgG scaffolds.

42. Use of a method, assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. And / or DPP3 activity is determined in the body fluid sample of the subject, said determination comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, use.

43. Use of a method, assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. And / or DPP3 activity is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is immobilized on the surface. use.

44. Use of a method, assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. The amount and / or DPP3 activity of the sample is determined in the body fluid sample of interest, and the separation step is a washing step of removing components of the sample that are not bound to the capture binder from the captured DPP3, use.

45. Use of a method, assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. Is determined in the body fluid sample of the subject, and the conversion of the substrate of DPP3 is the fluorescence of the fluorescent substrate (eg Arg-Arg-βNA, Arg-Arg-AMC), the color change of the color-developing substrate, and the substrate bound to aminoluciferin. Active staining (fixation) after luminescence (Promega's Protein-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis. Used, detected by a method selected from the group containing activated active DPP3), or western blots (cleavage products).

46. Use of a method, assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. Is determined in the body fluid sample of the subject, and the substrates are angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin, proctrin, ACTH and Use, selected from the group comprising MSH, or a dipeptide bound to a fluorescent group, chromophore or enkephalin (where the dipeptide is Arg-Arg).

47. Use of a method, assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of adverse events in said subject. Is determined in the body fluid sample of the subject and the substrate is selected from the group comprising a dipeptide bound to a fluorescent group, chromophore or aminoluciferin (where the dipeptide is Arg-Arg), use.

48. With the use of methods, assays or kits for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. There, the above method is
-The step of determining the level of DPP3 in the body fluid sample of the subject;
-With the process of comparing the determined DPP3 level with a predetermined threshold;
• Including the step of correlating the level of DPP3 with the risk of adverse events in the subject.
Use, where elevated levels above a certain threshold predict an increased risk of said organ dysfunction or deterioration of organ function.

49. With the use of methods, assays or kits for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. The use, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum.

50. With the use of methods, assays or kits for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein. use.

51. With the use of methods, assays or kits for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. Therefore, the method of determining the DPP3 activity in the body fluid sample of the subject is
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
Use, including the step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.

52. With the use of methods, assays or kits for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, and the determination comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody. Used, selected from the group of antibody fragments, or non-IgG scaffolds.

53. With the use of methods, assays or kits for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, and the determination comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody. ,use.

54. With the use of methods, assays or kits for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, and the determination comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is immobilized on the surface. Has been used.

55. 55. With the use of methods, assays or kits for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. A washing step in which the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of interest and the separation step removes components of the sample that are not bound to the capture binder from the captured DPP3. Is, use.

56. With the use of methods, assays or kits for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. Therefore, the DPP3 activity is determined in the body fluid sample of the subject, and the conversion of the substrate of DPP3 is the fluorescence of the fluorescent substrate (for example, Arg-Arg-βNA, Arg-Arg-AMC), the color change of the color-developing substrate, and aminoluciferin. After luminescence of the substrate bound to (Promega's Protein-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis. Active staining (immobilized active DPP3), or detected by a method selected from the group containing Western blots (cleavage products), used.

57. With the use of methods, assays or kits for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. DPP3 activity was determined in the body fluid sample of the subject, and the substrates were angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorfin. , Proctoline, ACTH and MSH, or selected from the group comprising dipeptides bound to fluoresceins, chromophores or aminoluciferins (where the dipeptide is Arg-Arg), use.

58. With the use of methods, assays or kits for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of said subject for the purpose of predicting the risk of organ dysfunction or deterioration of organ function in said subject. The DPP3 activity was determined in the body fluid sample of interest and the substrate was selected from the group comprising dipeptides bound to fluoresceins, chromophores or aminoluciferins (where the dipeptide is Arg-Arg). Use.

59. A method of predicting the risk of an adverse event in the subject, wherein the method is:
-The step of determining the level of DPP3 in the body fluid sample of the subject;
-With the process of comparing the determined DPP3 level with a predetermined threshold;
Including the step of correlating the level of DPP3 with the risk of the adverse event in the subject.
A method of predicting an increased risk of said adverse event by elevated levels above a certain threshold.

60. A method of predicting the risk of adverse events in a subject, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum.

61. A method of predicting the risk of adverse events in the subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the subject's body fluid sample and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein. Method.

62. A method of predicting the risk of adverse events in the subject, wherein DPP3 activity is determined in the subject's body fluid sample.
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
A method comprising the step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.

63. A method of predicting the risk of adverse events in the subject, in which the amount and / or DPP3 activity of the DPP3 protein is determined in the subject's body fluid sample, and the determination is a capture binder that specifically binds to full-length DPP3. A method in which the capture binder is selected from a group of antibodies, antibody fragments, or non-IgG scaffolds, comprising the use of.

64. A method of predicting the risk of adverse events in the subject, in which the amount and / or DPP3 activity of the DPP3 protein is determined in the subject's body fluid sample, and the determination is a capture binder that specifically binds to full-length DPP3. A method, wherein the capture binder is an antibody, comprising the use of.

65. A method of predicting the risk of adverse events in the subject, in which the amount and / or DPP3 activity of the DPP3 protein is determined in the subject's body fluid sample, and the determination is a capture binder that specifically binds to full-length DPP3. A method in which the capture binder is immobilized on the surface, including the use of.

66. A method of predicting the risk of adverse events in the subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the subject's body fluid sample and the separation step is not bound to the capture binder. A method, a cleaning step that removes components from captured DPP3.

67. A method of predicting the risk of adverse events in the subject, where DPP3 activity is determined in the subject's body fluid sample and conversion of the DPP3 substrate is performed by a fluorescent substrate (eg, Arg-Arg-βNA, Arg-Arg-AMC). ) Fluorescence, color change of color-developing substrate, luminescence of substrate bound to aminoluciferin (Promega's Protease-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer A method detected by a method selected from a group comprising chromatography, capillary zone electrophoresis, active staining after gel electrophoresis (immobilized active DPP3), or western blot (cut product).

68. A method of predicting the risk of adverse events in the subject, where DPP3 activity is determined in the subject's fluid sample and the substrates are angiotensin II, III, and IV, Leu-enkephalin, Met-enkephalin, endomorphin. Selected from groups containing 1 and 2, barolphin, β-casomorphin, dynorphin, proctrin, ACTH and MSH, or dipeptides bound to fluorinated groups, chromophores or aminoluciferins (where the dipeptide is Arg-Arg). The method.

69. A method for predicting the risk of adverse events in the subject, wherein the DPP3 activity is determined in the subject's body fluid sample and the substrate is a dipeptide with a fluorophore, chromophore or aminoluciferin attached (provided that the dipeptide is A method selected from a group containing (Arg-Arg).

70. A method for predicting the risk of organ dysfunction or deterioration of organ function in the subject.
-The step of determining the level of DPP3 in the body fluid sample of the subject;
-With the process of comparing the determined DPP3 level with a predetermined threshold;
• Including the step of correlating the level of DPP3 with the risk of adverse events in the subject.
A method in which an elevated level above a certain threshold predicts an increased risk of said organ dysfunction or deterioration of organ function.

71. A method of predicting the risk of organ dysfunction in a subject, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum.

72. A method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the subject's body fluid sample and:
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
A step of quantifying the DPP3 activity by adding a DPP3 substrate to the separated DPP3 and then measuring and quantifying the conversion of the DPP3 substrate, or a step of quantifying the amount of the DPP3 protein. Method.

73. A method for predicting the risk of organ dysfunction or deterioration of organ function in the subject, wherein the DPP3 activity is determined in the body fluid sample of the subject.
-The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
-Step of separating DPP3 bound to the capture binder,
-Step of adding the substrate of DPP3 to the separated DPP3,
A method comprising the step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3.

74. A method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the subject's body fluid sample, and the determination is specific for full-length DPP3. A method comprising the use of a capture binder that binds to, wherein the capture binder is selected from a group of antibodies, antibody fragments, or non-IgG scaffolds.

75. A method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the subject's body fluid sample and the determination is specific for full-length DPP3. A method comprising the use of a capture binder that binds to, wherein the capture binder is an antibody.

76. A method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the subject's body fluid sample and the determination is specific for full-length DPP3. A method comprising the use of a capture binder to bind to, wherein the capture binder is immobilized on a surface.

77. A method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, wherein the amount and / or DPP3 activity of the DPP3 protein is determined in the subject's body fluid sample and the separation step binds to the capture binder. A method, which is a cleaning step that removes the components of a sample that has not been captured from the captured DPP3.

78. A method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, where DPP3 activity is determined in the subject's body fluid sample and conversion of the DPP3 substrate is performed by a fluorescent substrate (eg, Arg-Arg-βNA, Arg-Arg-AMC) fluorescence, color change of color-developing substrate, luminescence of substrate bound to aminoluciferin (Promega Protease-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion) Chromatography), thin layer chromatography, capillary zone electrophoresis, active staining after gel electrophoresis (immobilized active DPP3), or detected by a method selected from the group comprising western blots (cleavage products). Method.

79. A method of predicting the risk of organ dysfunction or deterioration of organ function in the subject, where DPP3 activity is determined in the subject's body fluid sample and the substrates are angiotensin II, III, and IV, Leu-enkephalin, Met. -Enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or dipeptides bound to phosphors, chromophores or aminoluciferins (where the dipeptide is Arg-Arg). A method selected from the including groups.

80. A method for predicting the risk of organ dysfunction or deterioration of organ function in the subject, a dipeptide in which DPP3 activity is determined in the subject's body fluid sample and the substrate is attached to a fluorophore, chromophore or aminoluciferin. However, the method selected from the group comprising (where the dipeptide is Arg-Arg).

81. The treatment with angiotensin receptor agonists and / or precursors thereof is discontinued when the patient has an amount and / or DPP3 activity of DPP3 protein in a fluid sample below a predetermined threshold level. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 1 to 23.

82. The treatment with angiotensin receptor agonists and / or precursors thereof is initiated when the patient has an amount and / or DPP3 activity of DPP3 protein in the fluid sample above a predetermined threshold level, items 1-23. And angiotensin receptor agonists and / or precursors thereof for use in the treatment of the disease in the subject according to any one of paragraph 81.

Example 1: Method for measuring DPP3 protein and DPP3 activity

Antibody development and measurement of DPP3 binding capacity: Several mouse antibodies were generated and screened for binding capacity to human DPP3 by a special binding assay (see Table 1).

Peptides / conjugates for immunization

To conjugate with bovine serum albumin (BSA), an immunizing DPP3 peptide with an additional N-terminal cysteine residue (if cysteine is not present in the selected DPP3 sequence) was synthesized. (See Table 1, JPT Technologies, Berlin, Germany). The peptide was covalently attached to BSA using a Sulfolink coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was carried out according to the Perbio manual. Recombinant GST-hDPP3 was made by USBio (United States Biological, Salem, Mass., USA).

Mouse immunization, immune cell fusion and screening:

Intraperitoneal (i.p.) in Balb / c mice, 84 μg GT-hDDP3 (emulsified in TiterMax Gold adjuvant) or 100 μg DPP3-peptide-BSA-conjugate on day 0 (in complete Freund's adjuvant). 84 μg or 100 μg (emulsified with) was injected on day 14, and 42 μg or 50 μg (emulsified in an incomplete Freund's adjuvant) was injected on days 21 and 28. On day 49, mice were injected intravenously (i.v.) with 42 μg GT-hDDP3 or 50 μg DPP3-peptide-BSA-conjugate in saline. Three days later, the mice were euthanized and fused with immune cells.

1 ml of 50% polyethylene glycol was used at 37 ° C. for 30 seconds to fuse spleen cells from immunized mice with cells from the myeloma cell line SP2 / 0. After washing, cells were seeded on 96-well cell culture plates. Hybrid clones were selected in HAT medium [RPMI 1640 medium supplemented with 20% fetal bovine serum and HAT supplements]. After 1 week, the HAT medium was replaced with HT medium for 3 passages and then returned to normal cell medium.

Two weeks after fusion, screening for IgG antibodies that bind to recombinant DPP3 was first performed in cell culture supernatants. Therefore, GST-tagged recombinant hDPP3 (USBiologicals, Salem, USA) was immobilized on 96-well plates (100 ng / well) and incubated with 50 μl of cell culture supernatant per well for 2 hours at room temperature. After washing the plates, 50 μl / well of POD-rabbit anti-mouse IgG was added and incubated for 1 hour at room temperature. After the next washing step, 50 μl of chromophore solution (citrate / hydrogen phosphate buffer containing 3.7 mM o-phenylenediamine, 0.012% H ₂ O ₂ ) was added to each well and 15 at room temperature. After incubating for minutes, the color reaction was stopped by adding 50 μl of 4N sulfuric acid. Absorption was detected at 490 nm.

Microcultures positive for testing were transferred to 24-well plates and grown. After retesting, cloning and recloning of the selected cultures was performed using limiting dilution techniques to reveal the isotypes.

Preparation of mouse monoclonal antibody

Antibodies to GST-tagged human DPP3 or DPP3 peptides were prepared by standard antibody preparation methods (Marx et al. 1997) and purified through Protein A. The purity of the antibody was 90% or higher based on SDS gel electrophoresis analysis.

Antibody characterization-binding to hDPP3 and / or immunized peptides

Binding assays were performed to analyze the ability of various antibodies and antibody clones to bind to DPP3 / immunized peptides.

a) Solid phase

Highly bound microtitration plate (96-well polystyrene microplate, Greiner Bio-One international AG, Austria, Well 1) with GST-tagged recombinant hDPP3 (SEQ ID NO: 1) or DPP3 peptide (immunized peptide, SEQ ID NO: 2) Immobilized on the surface of a coupling buffer [50 mM Tris, 100 mM NaCl, pH 7.8] containing 1 μg per sap, 1 hour at room temperature). After blocking with 5% bovine serum albumin, the microplates were vacuum dried.

b) Signing procedure (tracer)

100 μg (100 μl) of various anti-DPP3 antibodies (1 mg / ml detection antibody in PBS, pH 7.4) to 10 μl of acridinium NHS ester (1 mg / ml in acetonitrile, InVent GmbH, Germany; EP It was mixed with 0 353 971) and incubated at room temperature for 30 minutes. The labeled anti-DPP3 antibody was purified by gel filtration HPLC on Shodex Protein 5 μm KW-803 (Showa Denko, Japan). Purified labeled antibody in an assay buffer (50 mmol / l potassium phosphate, 100 mmol / l NaCl, 10 mmol / l Na ₂ -EDTA, 5 g / l bovine serum albumin, 1 g / l Diluted in mouse IgG, 1 g / l bovine IgG, 50 micromolar / l amasstatin, 100 micromolar / l leupeptin, pH 7.4). The final concentration was about 5-7 × 10 ⁶ relative light units (RLU) per 200 μl of labeled compound (about 20 ng of labeled antibody). The chemiluminescence of the acridinium ester was measured using a Centro LB 960 luminescence measuring device (Berthold Technologies GmbH & Co. KG).

c) hDPP3 binding assay

Plates were filled with 200 μl of labeled and diluted detection antibody (tracer) and incubated at 2-8 ° C. for 2-4 hours. The unbound tracer was removed by washing 4 times with 350 μl of wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Chemiluminescence of well-bound antibody was measured using a Centro LB 960 luminescence meter (Berthold Technologies GmbH & Co. KG).

Antibody characterization-hDPP3 inhibitory analysis

A DPP3 activity assay was performed using a known procedure (Jones et al., 1982) to analyze the ability of various antibodies and antibody clones to suppress DPP3. GST-tagged recombinant hDPP3 was diluted in assay buffer (50 mM Tris-HCl, 25 ng / ml GST-DPP3 in pH 7.5 and 100 μM ZnCl ₂ ) and 200 μl of this solution with 10 μg of each antibody. Incubated at room temperature. After 1 hour of pre-incubation, the fluorescent substrate Arg-Arg-βNA (20 μl, 2 mM) was added to this solution and free βNA using a Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG). Generation was monitored at 37 ° C. Fluorescence of βNA is detected by excitation at 340 nm and measuring luminescence at 410 nm. Calculate the increasing fluorescence gradient (in RFU / min) for different samples. A gradient of natural human DPP3 using a buffer control is 100% active. Possible inhibitory activity of the capture binder is defined as a decrease in GST-hDPP3 activity (in%) by incubation with the capture binder.

The table below shows the obtained series of antibodies and their binding rate (unit is relative optical unit (RLU)), as well as relative inhibitory ability (%; Table 1). Monoclonal antibodies against the DPP3 region below were selected based on their ability to bind recombinant DPP3 and / or immunized peptides and their ability to suppress them.

All antibodies to the GST-tagged full-length form of recombinant hDPP3 showed strong binding to immobilized GST-tagged hDPP3. Antibodies to the peptide of SEQ ID NO: 2 also bind to GST-hDPP3. The antibody of SEQ ID NO: 2 also binds strongly to the immunized peptide.

表1：完全長hDPP3またはhDPP3の配列に対する抗体と、hDPP3（配列番号1）または免疫化ペプチド（配列番号2）への結合能力（単位はRLU）のほか、組み換えGST- hDPP3の最大抑制率のリスト

Table 1: Antibodies to full-length hDPP3 or hDPP3 sequences and ability to bind hDPP3 (SEQ ID NO: 1) or immunized peptide (SEQ ID NO: 2) (unit: RLU), as well as maximum inhibition of recombinant GST-hDPP3. list

The development of a fluorescent immunoassay (DPP3-LIA) for quantifying the concentration of DPP3 protein and an enzyme capture activity assay (DPP3-ECA) for quantifying DPP3 activity was recently reported (Rehfeld et al. 2018 JALM printing). .. This article is incorporated herein by reference in its entirety.

Example 2: DPP3 for predicting early mortality

The hDPP3 immunoassay was used to determine plasma DPP3 levels in patients with a variety of diseases and to correlate them with early mortality.

Study Cohort-Sepsis and Septic Shock

Plasma samples from 574 patients from studies of adrenomedulin and outcomes in severe sepsis and septic shock (AdrenOSS) were screened for DPP3. AdrenOSS is a prospective observational multinational study involving 583 patients enrolled in the intensive care unit with sepsis or septic shock (Hollinger et al., 2018). 292 patients were diagnosed with septic shock.

Research Cohort-Cardiogenic Shock

Plasma samples from 108 patients diagnosed with cardiogenic shock were screened for DPP3. Blood was collected within 6 hours of detection of cardiogenic shock. Death was followed for 7 days.

Study Cohort-Acute Coronary Syndrome

Plasma samples from 720 patients with acute coronary syndrome were screened for DPP3. Blood was drawn 24 hours after the onset of chest pain. Death was followed for 7 days.

Study Cohort-Dyspnea

Plasma samples were collected from 1440 patients with dyspnea (shortness of breath) shortly after admission to the emergency department of Skane University Hospital. Patients with dyspnea may be at increased risk of organ failure and premature death, especially because they may have acute coronary syndrome or congestive heart failure. Death was followed for 3 months after appearing in the emergency department.

Study Cohort-Burns Patients

107 patients with severe burns (> 15% of total body surface area) were screened for DPP3. Blood was collected at admission. Death was followed for 4 weeks.

hDPP3 immunoassay:

The level of DPP3 contained in the patient's plasma was determined using an immunoassay (LIA) that detects the amount of human DPP3 or an activity assay (ECA) that detects the activity of human DPP3. Antibody immobilization, labeling, and incubation were performed as described by Rehfeld et al. (Rehfeld et al. 2018).

result

Short-term patient survival in sepsis / septic shock was associated with DPP3 plasma levels at admission. Patients with DPP3 plasma levels above 68.6 ng / ml (three quartiles) had an increased risk of dying compared to patients with DPP3 plasma levels below this threshold (Figure 1A). The same relationship was found when the relationship between short-term outcomes and DPP3 plasma levels was analyzed only in patients with septic shock in this cohort (Fig. 1F). Patients with elevated DPP3 plasma levels had an increased risk of death compared to patients with low DPP3 plasma levels. When the same cutoff value is applied to patients with cardiogenic shock, it is also observed that patients with high DPP3 have an increased risk of premature death within 7 days (Fig. 1B).

In addition, 28-day survival in patients with acute coronary syndrome associated with DPP3 is also lower when DPP3 is large when applying the respective cutoff values of 68.6 ng / ml (Fig. 1C).

Applying this cutoff value of 68.6 ng / ml to patients suffering from dyspnea detected a significant increased risk of death in patients with high DPP3 during a 3-month follow-up period (Figure 1D).

In addition, patients with severe burns with high DPP3 concentrations above their respective cutoff values of 68.6 ng / ml had an increased risk of 4-week death (Fig. 1E).

Example 3: Purification of human natural DPP3

A total of 100 ml of human erythrocyte lysate was applied to Sepahrose 4B resin (Sigma-Aldrich) and the flow-through was recovered. The resin was washed with a total of 370 ml of PBS buffer, pH 7.4 and combined with the flow-through from which the washed fraction was recovered to give a total volume of 2370 ml.

For the immunoaffinity purification process, 110 mg of monoclonal anti-hDPP3 mAb AK2552 was coupled to 25.5 ml of UltraLink hydrazide resin (Thermo Fisher Scientific) according to the manufacturer's (GlycoLink Immobilization Kit, Thermo Fisher Scientific) protocol. The coupling efficiency determined by quantifying uncoupled antibodies by Bradford technique was 98%. Equilibrate the resin-antibody conjugate in a wash-binding buffer (PBS, 0.1% Triton X-100, pH 7.4) 10 times the volume of the floor, combine with 2370 ml of clear red blood cell lysate and stir continuously. Incubated at 4 ° C for 2 hours. As a result, a 100 ml incubation mixture spread on the surface of 10 15 ml polypropylene columns and was recovered by centrifuging the flow-through at 1000 xg for 30 seconds. By repeating this process several times, a resin loaded with 2.5 ml of DPP3 per column was obtained. Each column was washed 5 times with 10 ml wash-binding buffer using a gravity-glow approach. After elution of DPP3 by placing each column in a 15 ml falcon tube containing 2 ml of neutralization buffer (1 M Tris-HCl, pH 8.0), a 10 ml elution buffer per column (1 M Tris-HCl, pH 8.0). 100 mM glycine-HCl, 0.1% Triton X-100, pH 3.5) was added, and immediately after that, the mixture was centrifuged at 1000 × g for 30 seconds. This elution step was repeated 3 times to give a total of 360 ml of eluent. The pH of the neutralized eluent was 8.0.

5 ml HiTrap of the combined eluents equilibrated with IEX buffer A1 (100 mM glycine, 150 mM Tris, pH 8.0) using a sample pump from the Akta Start system (GE Healthcare). Loaded into a Q-sephare HP column (GE Healthcare). After loading the sample, the column was washed with 5 times the volume of IEX buffer A2 (12 mM NaH ₂ PO ₄ , pH 7.4) to remove unbound protein. Elution of DPP3 is sodium chloride in the range 0-1 M in 10 times the volume (50 ml) of the column using IEX buffer B (12 mM NaH ₂ PO ₄ , 1 M NaCl, pH 7.4). This was achieved by applying a gradient. The eluate was collected in 2 ml fractions. The buffer used for ion exchange chromatography was sterilized and filtered using a 0.22 μM bottle-top filter.

Table 2 shows a purification table showing the yield and activity of each purification step. FIG. 2 shows SDS-PAGE on a gradient gel (4-20%) for native hDPP3 purified from human erythrocyte lysate.

a）DPP3-LIAアッセイを利用してDPP3の相対量をすべての分画で求めた。出発材料に含まれるDPP3の量を100％に設定し、精製分画に含まれる残留DPP3の量を出発材料と相関させた。
b）全タンパク質の量は、Petersonによって改変されたLowryの方法を利用して求めた（Peterson 1977年 Analytical Biochemistry 第356巻：346～356ページ）。
c）1分当たりの変換された基質の全Arg₂-βNA加水分解活性（単位はマイクロモル）は、Dpp3-ECAを利用して求め、β-ナフチルアミン（0.05～100μM）を通じて較正した。
d）精製の収率は、全Arg₂-βNA加水分解活性から計算した。出発材料に含まれるArg₂-βNA加水分解活性を100％に設定した。
e）比活性は、全タンパク質1 mg当たりの1分間で変換された基質のマイクロモルと定義する。
f）精製因子は、各精製工程の後と前の比活性の商である。

a) The relative amount of DPP3 was determined in all fractions using the DPP3-LIA assay. The amount of DPP3 contained in the starting material was set to 100% and the amount of residual DPP3 contained in the purified fraction was correlated with the starting material.
b) The amount of total protein was determined using Lowry's method modified by Peterson (Peterson 1977 Analytical Biochemistry Vol. 356: pp. 346-356).
c) Total Arg ₂ -βNA hydrolysis activity (in micromol) of the converted substrate per minute was determined using Dpp3-ECA and calibrated through β-naphthylamine (0.05-100 μM).
d) Purification yields were calculated from total Arg ₂ -βNA hydrolysis activity. The Arg ₂ -βNA hydrolysis activity contained in the starting material was set to 100%.
e) Specific activity is defined as micromol of substrate converted in 1 minute per 1 mg of total protein.
f) Purification factors are the quotient of specific activity after and before each purification step.

Example 4: Effect of Natural DPP3 on Animal Models

The effect of injecting natural hDPP3 into healthy mice was investigated by monitoring the inner diameter shortening rate.

Wild-type Black 6 mice (8-12 weeks old, see Table 3 for group size) were acclimatized for 2 weeks before performing baseline echocardiography. Mice were randomly assigned to one of the two groups and then intravenously injected with native DPP3 protein or PBS posteriorly at a dose of 600 μg per kg of DPP3 protein.

Cardiac function was assessed by echocardiography (Gao et al., 2011) at 15, 60, and 120 minutes after injection of DPP3 or PBS, and renal function was assessed by renal resistance index (Lubas et al. 2014; Dewitte). Others 2012) (Fig. 3).

表3：実験群のリスト

Table 3: List of experimental groups

result

Mice treated with the native DPP3 protein show a significant reduction in inner diameter shortening rate compared to the PBS-injected control group (Fig. 4A). The wild-type + DPP3 group also showed deterioration of renal function, as evidenced by the increase in renal resistance index (Fig. 4B).

Example 5: Development of Procizumab

Antibodies to SEQ ID NO: 2 were characterized in more detail (epitope mapping, binding affinity, specificity, inhibitory capacity). Here, the result of clone 1967 (AK1967; "Procizumab") of SEQ ID NO: 2 is shown as an example.

Determination of AK1967 epitope for DPP3

To map the epitope of AK1967, a number of N-terminal or C-terminal biotinylated peptides were synthesized (peptides & elephants GmbH, Hennigsdorf, Germany). These peptides contain the sequence of a fully immunized peptide (SEQ ID NO: 2), or a fragment thereof from which one amino acid has been removed from the C-terminus or N-terminus (see Table 5 for a complete list of peptides). I want to be).

Highly bound 96-well plates were coated with a coupling buffer (500 mM Tris-HCl, pH 7.8, 100 mM NaCl) containing 2 μg of avidin (Greiner Bio-One international AG, Austria) per well. .. The plates were then washed and filled with a special solution of biotinylated peptide (10 ng / well; buffer-1 x PBS with 0.5% BSA).

The anti-DPP3 antibody AK1967 was labeled with a chemiluminescent label according to Example 1.

Plates were filled with labeled and diluted 200 μl of detection antibody (tracer) and incubated at room temperature for 4 hours. The unbound tracer was removed by washing 4 times with 350 μl of wash solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). The chemiluminescence of a well-bound antibody was measured using a Centro LB 960 luminescence meter (Berthold Technologies GmbH & Co. KG). Binding of AK1967 to each peptide was determined by assessing the relative light unit (RLU). Any peptide whose RLU signal is significantly greater than the non-specific binding of AK1967 is defined as the AK1967 binder. Combinatorial analysis of binding and non-binding peptides reveals a specific DPP3 epitope of AK1967.

Measurement of binding affinity using Octet:

Experiments were performed using Octet Red96 (ForteBio). AK1967 was captured on a kinetic grade anti-human Fc (AHC) biosensor. The loaded biosensor was then immersed in a dilution sequence of GSR-tagged recombinant human DPP3 (100, 33.3, 11.1, 3.7 nM). After observing the association for 120 seconds, the dissociation was observed for 180 seconds. Table 4 shows the buffers used in the experiment. A dynamic analysis was performed using a 1: 1 binding model and global fitting.

表4：Octet測定のために使用したバッファ

Table 4: Buffers used for Octet measurements

Western blot analysis of binding specificity of AK1967

Blood cells from human EDTA-blood were washed (three times in PBS), diluted in PBS and lysed by repeating the dry-freeze cycle. This blood cell lysate had a total protein concentration of 250 μg / ml and a DPP3 concentration of 10 μg / ml. SDS-PAGE and Western blot with diluted blood cell lysate (1:40, 1:80, 1: 160, and 1: 320) and purified recombinant human His-DPP3 diluted (31.25-500 ng / ml). Was carried out. Blots were placed in 1) blocking buffer (1 x PBS-T with 5% skim milk powder), 2) in primary antibody solution (AK1967, 1: 2,000 in blocking solution), and 3) in HRP. Incubated in labeled secondary antibody (goat anti-mouse IgG, 1: 1,000 in blocking solution). Bound secondary antibodies were detected using Amersham ECL Western blotting detection reagents and Amersham Imager 600 UV (both from GE Healthcare).

DPP3 suppression assay:

To analyze the ability of AK1967 to suppress DPP3, a DPP3 activity assay was performed using a known procedure (Jones et al., 1982) as described in Example 1. The inhibitory capacity of AK1967 is defined as a decrease in GST-hDPP3 activity by incubation with the antibody (unit:%). The resulting decrease in DPP3 activity is shown in the suppression curve in Figure 1C.

Epitope mapping:

Analysis of peptides that bind to AK1967 and peptides that do not bind to AK1967 revealed the DPP3 sequence INPETG (SEQ ID NO: 3) as an epitope required for binding to AK1967 (see Table 5).

表5：AK1967のエピトープマッピングに用いたペプチド

Table 5: Peptides used for epitope mapping of AK1967

Binding affinity:

AK1967 binds to recombinant GST-hDPP3 with an affinity of 2.2 × 10 ^-9 M (see dynamic curve in Figure 5).

Specificity and suppressive ability:

The only protein detected using AK1967 as the primary antibody in blood cell lysates was DPP3 at the 80 kDa position (Fig. 6). The total protein concentration of lysate was 250 μg / ml, while the estimated DPP3 concentration was about 10 μg / ml. There were 25 times more non-specific proteins in the lysate, but AK1967 specifically binds to DPP3 and detects DPP3, with no other non-specific binding.

AK1967 suppresses 15 ng / ml DPP3 in a specific DPP3 activity assay, with an IC ₅₀ of approximately 15 ng / ml (Fig. 7).

Chimerization / humanization:

The monoclonal antibody AK1967 (“Procizumab”), which has the ability to suppress DPP3 activity by 70%, was selected as a possible therapeutic antibody and also used as a template for chimerization and humanization.

Humanization of mouse antibodies can be performed according to the following procedure:

To humanize antibodies of mouse origin, antibody sequences are analyzed for structural interactions of antigens with framework regions (FRs) that have complementarity determining regions (CDRs). Appropriate FRs of human origin are selected based on structural modeling and mouse CDR sequences are transplanted into human FRs. Variations can be introduced into the amino acid sequence of CDR or FR to regain structural interactions. This structural interaction was disabled by the seed switch for the FR sequence. This recovery of structural interactions can be achieved by a random approach using phage presentation libraries or through a direct approach guided by molecular modeling (Almagro and Fransson, 2008 "Humanization of Antibodies" Front Biosci. Volume 13: pp. 1619-33).

In the above context, variable regions can be connected to any subclass of constant regions (IgG, IgM, IgE, IgA), or only to scaffolds, Fab fragments, Fv, Fab, and F (ab) ₂ . can. In Examples 6 and 7 below, a mouse antibody variant having an IgG2a skeleton is used. A human IgG1κ skeleton was used for chimerization and humanization.

Only complementarity determining regions (CDRs) are important for epitope binding. CDRs for heavy and light chains of mouse anti-DPP3 antibody (AK1967; "procizumab") in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 for heavy chains and SEQ ID NO: 9, SEQ ID NO: 9 for light chains. It is shown in KVS and SEQ ID NO: 10, respectively.

Sequencing of the anti-DPP3 antibody (AK1967; "procizumab") revealed an antibody heavy chain variable region (H chain) according to SEQ ID NO: 11 and an antibody light chain variable region (L chain) according to SEQ ID NO: 12.

Example 6: Effect of Procizumab on Sepsis-Induced Heart Failure

In this experiment, the effect of injecting procizumab into sepsis-induced heart failure rats (Rittirsch et al., 2009) was investigated by monitoring the rate of inner diameter shortening.

CLP model of septic shock:

Male Wistar rats from Center d'elevage Janvier (France) (2-3 months old, 300-400 g, see Table 6 for group size) were randomly assigned to one of the three groups. All rats were anesthetized with ketamine hydrochloride (90 mg / kg) and xylazine (9 mg / kg) intraperitoneally (i.p.). Cecal ligation and perforation (CLP) was performed with a slight modification of Rittirsch's protocol to induce polybacterial sepsis. A ventral midline incision (1.5 cm) was performed to expose the cecum. The cecum was then connected just below the ileocecal valve and pierced once with an 18 gauge needle. The abdominal cavity is then closed in two layers, followed by fluid resuscitation (subcutaneous injection of 3 ml saline per 100 g of body weight) and the rat returned to its original cage. Surgery Surgery on rats, but no perforation of the cecum. CLP rats were randomly divided into a placebo group and a therapeutic antibody group.

Research design:

The flow of research is shown in Fig. 8. After CLP or sham surgery, rats were rested for 20 hours with free access to water and food. The rats were then anesthetized and a tracheostomy was performed to secure arterial and venous lines. Twenty-four hours after CLP surgery, AK1967 or vehicle (saline) was administered as a bolus injection at 5 mg / kg and then infused at 7.5 mg / kg for 3 hours. As a safety indicator, hemodynamics were invasively and continuously monitored from t = 0 to 3 hours later.

At t = 0 (baseline), all CLP rats were in septic shock with progressive decline in cardiac function (hypotension, small inner diameter shortening rate). At this point, procizumab or vehicle (PBS) was injected (i.v.) and saline infusion was started. There is one control group and two CLP groups, which are summarized in the table below (Table 6). Rats were euthanized at the end of the experiment and organs were harvested for later analysis.

表6：実験群のリスト

Table 6: List of experimental groups

Invasive blood pressure:

Hemodynamic variables were acquired using the AcqKnowledge system (BIOPAC Systems, Inc., USA). This system provides a fully automated blood pressure analysis system. Connect the catheter to the BIOPAC system via a pressure sensor.

Rats were anesthetized to proceed with the procedure (ketamine and xylazine). Rats were moved to a heating pad to the desired body temperature of 37-37.5 ° C. A temperature feedback probe was inserted into the rectum. The rat was placed on the workbench in the supine position. An incision was made in the trachea for external ventilation and a catheter (16G) was inserted without damaging the carotid artery and vagus nerve. An arterial catheter was inserted into the right carotid artery. Separate the carotid artery from the vagus nerve prior to ligation.

A central venous catheter was inserted through the left jugular vein to allow administration of PCZ or PBS.

After the operation, the rats were rested in a stable state, and then the hemodynamics were measured. Baseline blood pressure (BP) was then recorded. Infusion of saline through the arterial line was stopped while the data was being collected.

Echocardiography:

Rats were anesthetized with ketamine hydrochloride. The chest was shaved and the rat was placed in a recumbent position.

A commercially available GE Healthcare Vivid 7 ultrasound system with a radio frequency (14-MHz) linear probe and a 10-MHz cardiac probe was used for transthoracic echocardiography (TTE) examination. All tests were digitally recorded and saved for later analysis offline.

Gray scale images were recorded at a depth of 2 cm. A two-dimensional examination was started on the parasternal axis and the diameter of the aortic annulus and the diameter of the pulmonary artery were measured. The size of the left ventricle (LV) was measured using M mode as well, and the inner diameter shortening rate (FS%) was evaluated. LVFS was calculated as LVend diastolic diameter-LVend systolic diameter / LVend diastolic diameter and expressed in%. Therefore, the time point at the end of diastole was defined as the time point at which the LV had the maximum diameter. Therefore, the time point at the end of systole was defined as the time point with the smallest diameter in the same heartbeat cycle. All parameters were measured manually. Three heart rate cycles were averaged for each measurement.

Pulmonary artery flow was recorded using a pulse wave Doppler from the same parasternal long axis. The velocity-time integral of the pulmonary artery outflow was measured.

Trapezius flow was recorded at the tip level of the mitral valve using a pulsed Doppler from the apex of the heart.

result:

Treatment of sepsis-induced heart failure rats with PBS (CLP + PBS) reduced the rate of inner diameter shortening compared to sham-surgery rats (Fig. 9A). The CLP + PBS group also showed a high mortality rate (Fig. 9B). In contrast, application of procizumab to sepsis-induced heart failure rats improved the rate of inner diameter shortening (Fig. 9A) and dramatically reduced mortality (Fig. 9B).

Example 7: Effect of Procizumab on Heart Failure and Renal Failure

The effect of procizumab on isoproterenol-induced heart failure in mice was investigated by monitoring the inner diameter shortening rate and the renal resistance index.

Isoproterenol-induced cardiac stress in mice:

Isoproterenol (DL-isoprenaline hydrochloride, Sigma Chemical Co.) (ISO), a non-selective β-adrenergic agonist, was given to 3-month-old male mice at 300 mg / kg twice daily, 2 Subcutaneous injection over a day induced acute heart failure (Vergaro et al., 2016). ISO was diluted in NaCl 0.9%. Mice treated with isoproterenol were randomly assigned to the two groups (Table 7), and after baseline echocardiography (Gao et al., 2011), PBS or procizumab (10 mg / kg) was injected intravenously. Renal resistance index measurement (Lubas et al. 2014; Dewitte et al. 2012) was performed on the third day (Fig. 10A and Fig. 10B).

Echocardiography (Gao et al. 2011) and renal resistance index (Lubas et al. 2014; Dewitte et al. 2012) were used to assess cardiac function at 1 hour, 6 hours, and 24 hours (Fig. 10A). And Figure 10B). Mice in the group injected with vehicle (PBS) instead of isoproterenol received no further drug treatment and were used as a control group (Table 7).

表7：実験群のリスト

Table 7: List of experimental groups

result:

Application of procizumab to isoproterenol-induced heart failure mice recovers from heart failure within the first hour of administration (Fig. 11A). The renal function of diseased mice showed a significant improvement 6 hours after injection of PCZ and was comparable to that of sham-operated rats at 24 hours (Fig. 11B).

Example 8: Effect of Valsartan

The effect of balsartan, an antagonist of the type 1 angiotensin II receptor (ATR1), was investigated by monitoring the inner diameter shortening rate in healthy mice injected with DPP3.

In this experiment, healthy Black 6 mice (8-12 weeks old, see Table 8 for group size) consumed water containing 50 mg / kg valsartan per day, or water alone over a two-week period. (Table 8). Both groups were then intravenously injected with natural DPP3 (600 μg / kg) and the inner diameter shortening rate was evaluated according to Gao et al., 2011 at 15 minutes, 60 minutes and 120 minutes (Fig. 12).

表8：実験群のリスト

Table 8: List of experimental groups

result:

Injection of DPP3 into healthy mice significantly reduced the rate of inner diameter shortening (Fig. 13). In contrast, healthy mice injected with DPP3 after treatment with the angiotensin II receptor agonist balsartan showed no signs of cardiac dysfunction as assessed by the rate of inner diameter shortening. Therefore, DPP3-mediated cardiac dysfunction is mediated by angiotensin II, as cardiac function was restored by treatment with valsartan.

Mice treated with balsartane for 2 weeks were adapted for inhibition of type 1 angiotensin II receptors, followed by angiotensin II-mediated signaling inhibition and AngII-mediated inhibition of cardiac function. .. Apparently, under balsartane treatment, mice switched to a different method of activating cardiac function, independent of type I angiotensin II receptor signaling. This is because this angiotensin signaling system was suppressed by valsartan.

When DPP3 cleaves Ang II and thus suppresses the activity of angiotensin II-mediated cardiac function, mice adapted to the down-regulated angiotensin system (valsartan-treated mice) are assessed by inner diameter shortening rate. There were no signs of cardiac dysfunction. In contrast, mice that were not treated with the angiotensin II receptor blocker balsartan and did not adapt to AngII-mediated signaling inhibition responded to DPP3 injection followed by AngII cleavage and inactivation. It showed a significant decrease in the inner diameter shortening rate.

This experiment underscores the relationship between DPP3 and angiotensin II, implying that DPP3-induced heart failure is mediated by angiotensin II.

Example 9: DPP3 and organ dysfunction in sepsis

Using the same study described in Example 2 (AdrenOSS-1), cDPP3 and organs of patients hospitalized for sepsis and septic shock (eg, cardiovascular and renal dysfunction). Evaluated the relationship between them. AdrenOSS-1 is a European observational multinational study involving 583 patients admitted to the ICU for sepsis or septic shock (ClinicalTrials.gov NCT02393781). The first result (as described in Example 2) was 28-day mortality. The second result included organ failure as defined by the SOFA score, organ support focused on the use of vasopressors, and the need for renal replacement therapy. Blood for the central laboratory was sampled within 24 hours and 2 days after being placed in the ICU.

The median cDPP3 measured on admission in all Adren OSS-1 patients was 45.1 ng / ml (interquartile range 27.5-68.6). High levels of DPP3 measured on admission were associated with worst metabolic parameters, renal and cardiac function, and SOFA score. Patients with median DPP3 levels have SOFAs compared to those with median DPP3 levels above the median 45.1 ng / ml with a median SOFA score of 8 (IQR 5-11). The median score (points) was 6 (IQR 4-9) (Fig. 14).

High levels of cDPP3 after 24 hours, regardless of cDPP3 levels at admission, were associated with the worst SOFA scores, both overall (Fig. 15) and organ-by-organ (Fig. 16A-16F).

Taken together, these data showed that high levels of cDPP3 were associated with survival and the degree of organ failure in patients with sepsis or septic shock in a large international cohort. The study found a significant relationship between cDPP3 <45.1 ng / ml at admission and short-term survival in both sepsis and septic shock, as well as a cutoff value of 45.1 ng / ml that predicts prognosis. Was done. For organ dysfunction, there was a positive relationship between cDPP3 and SOFA score at ICU admission. More importantly, the relationship between the level of cDPP3 and the degree of organ dysfunction seen during ICU containment also applies during the recovery phase. In fact, patients with high levels of cDPP3 at admission but diminished towards normal cDPP3 levels on day 2 are more likely to recover function in all organs, including cardiovascular, kidney, lung, and liver. It was big.

arrangement

SEQ ID NO: 1-hDDP3 Amino Acids 1-737
MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSPEAPYIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTKFVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTYFSGNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDSEVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESFTQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFEGFVAVVNKAMSAKFERLVASAEQLLKELPWPPTFEKDKFLTPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEGFKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGKLFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLYLCLHPQVLEIFGFEGADAEDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARFVILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTGDVAGGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEYEASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA

SEQ ID NO: 2-hDDP3 Amino Acids 474-493 (N-Cys) -Immune Peptide with Additional N-Terminal Cysteine
CETVINPETGEQIQSWYRSGE

SEQ ID NO: 3 --hDDP3 Amino Acids 477-482 --Epitope of AK1967
INPETG

SEQ ID NO: 4-Variable regions within the heavy chain of mouse AK1967
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDNKSYNPALKSRLTISRDTSNNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS

SEQ ID NO: 5-Variable regions within the light chain of mouse AK1967
DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

SEQ ID NO: 6-CDR1 in the heavy chain of mouse AK1967
GFSLSTSGMS

SEQ ID NO: 7-CDR2 in the heavy chain of mouse AK1967
IWWNDNK

SEQ ID NO: 8-CDR3 in the heavy chain of mouse AK1967
ARNYSYDY

SEQ ID NO: 9-CDR1 in the light chain of mouse AK1967
RSLVHSIGSTY

CDR2 of the light chain of mouse AK1967
KVS

SEQ ID NO: 10-CDR3 in the light chain of mouse AK1967
SQSTHVPWT

SEQ ID NO: 11 --Humanized AK1967 --Heavy chain sequence (IgG1κ skeleton)
MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMDPVDTGTYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO: 12 --Humanized AK1967 --Light chain sequence (IgG1κ skeleton)
METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLYWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKTSVVC.

SEQ ID NO: 13-Angiotensin II (also known as 5-isoleucine-angiotensin II)
DRVYIHPF

SEQ ID NO: 14-Angiotensin II analog (5-valine-angiotensin II)
DRVYVHPF

SEQ ID NO: 15 --Angiotensin II analog (Asn ¹ -Phe ⁴ )
NRVFIHPF

SEQ ID NO: 16-Angiotensin II hexapeptide
VYIHPF

SEQ ID NO: 17-Angiotensin II nonapeptide
NRVYYVHPF

SEQ ID NO: 18-Angiotensin II analog ([Asn ¹ -Ile ⁵ -Ile ⁸ ]-Angiotensin II)
NRVYIHPI

SEQ ID NO: 19-Angiotensin II analog ([Asn ¹ -Ile ⁵ -Ala ⁸ ]-Angiotensin II)
NRVYIHPA

SEQ ID NO: 20-Angiotensin II analog ([Asn ¹ -Gindo Tyr ⁴ -Ile ⁵ ]-Angiotensin II)
NRVYIHPF

SEQ ID NO: 21-Angiotensin III
RVYIHPF

SEQ ID NO: 22-Angiotensin III analog (Val ⁴ -Angiotensin III)
RVYVHPF

SEQ ID NO: 23-Angiotensin III analog (Phe ³ -Angiotensin III)
RVFIHPF

SEQ ID NO: 24-Angiotensin III analog ([Ile ⁴ -Ala ⁷ ]-angiotensin III)
RVYIHPA

SEQ ID NO: 25-Angiotensin III analog ([Gindo Tyr ³ -Ile ⁴ ] angiotensin III)
RVYIHPF

SEQ ID NO: 26-Angiotensin IV
VYIHPF

SEQ ID NO: 27-Angiotensin IV analog (Val ³ -Angiotensin IV)
VYVHPF

SEQ ID NO: 28-Angiotensin IV analog (Phe ² -Angiotensin IV)
VFIHPF

SEQ ID NO: 29-Angiotensin IV analog ([Ile ³ -Ala ⁶ ]-Angiotensin IV)
VYIHPA

SEQ ID NO: 30-Angiotensin IV analog ([Gindo Tyr ² -Ile ³ ]-Angiotensin IV)
VYIHPF

Kaplan-Meier survival curves associated with low DPP3 plasma concentrations (<68.6 ng / ml) and high DPP3 plasma concentrations (≥68.6 ng / ml). (A) Relationship between 7-day survival rate and DPP3 plasma concentration in patients with septicemia / septic shock (cutoff value is 68.6 ng / ml); (B) 7-day survival rate and DPP3 in patients with cardiogenic shock Relationship between plasma concentration (cutoff value is 68.6 ng / ml); (C) Relationship between 7-day survival rate and DPP3 plasma concentration in patients with acute myocardial infarction (cutoff value is 68.6 ng / ml); (D) Respiration Relationship between 3-month survival rate and DPP3 plasma concentration in patients with difficulty; (E) Relationship between 4-week survival rate and DPP3 plasma concentration in burned patients . SDS-PAGE on a gradient gel (4-20%) for natural hDPP3 purified from human erythrocyte lysate. Molecular weight markers are indicated by arrows. Experimental design-Effects of natural hDPP3 in animal models. (A) Injection of DPP3 causes a decrease in the inner diameter shortening rate, which leads to deterioration of cardiac function. (B) Decreased renal function is also observed through the increased renal resistance index. Association-dissociation curve of AK1967-DPP3 binding analysis using Octet. Biosensors loaded with AK1967 were immersed in GST-tagged recombinant human DPP3 serial dilutions (100, 33.3, 11.1, 3.7 nM) to monitor dissociation and association. Western blotting of diluted blood cell lysate and detection of DPP3 using AK1967 as the primary antibody. Suppression curve in which natural DPP3 from blood cells was suppressed using the inhibitory antibody AK1967. The suppression of DPP3 by specific antibodies was concentration-dependent, with an IC50 of approximately 15 ng / ml when analyzed against 15 ng / ml DPP3. Experimental settings-The effect of procizumab on sepsis-induced heart failure. Procizumab dramatically improves inner diameter shortening rate (A) and mortality rate (B) in mice in which heart failure is induced by sepsis. Experimental settings-After inducing cardiac stress with isoproterenol in mice, treat with procizumab (B) and control (A). In mice in which heart failure was induced by isoproterenol, procizumab improved the inner diameter shortening rate within 1 hour after administration (A) and decreased the renal resistance index within 6 hours (B). Experimental settings-Effect of valsartan in healthy mice injected with DPP3. The reduction in inner diameter shortening rate due to DPP3 is relieved by valsartan treatment. High DPP3 concentration levels 24 hours after admission of septic patients were associated with the worst SOFA score. High cDPP3 plasma levels correlate with organ dysfunction in patients with sepsis. Bar graph of SOFA score obtained from changes in DPP3 level during ICU stay in Adren OSS1. HH: DPP3 greater than median at admission and 24 hours later; HL: greater than median at admission but less than median at 24 hours; LL: at admission and 24 hours later Below median; LH: below median at admission, but greater than median at 24 hours. High levels of cDPP3 levels 24 hours after admission of septic patients were associated with the worst SOFA score per organ. Fluctuating cDPP3 levels from admission to 24 hours after admission for (A) heart, (B) kidney, (C) respiratory, (D) liver, (E) coagulation, and (F) central nervous system SOFA score values (HH: high / high, HL: high / low, LH: low / high, LL: low / low).

Claims (60)

Angiotensin receptor agonist and / or precursor thereof for use in the treatment of disease in a subject.
The diseases include heart failure, chronic heart failure, acute heart failure (AHF), myocardial infarction (MI), stroke, liver failure, burns, trauma, severe infections (microbial disease, viral disease (eg AIDS), parasite disease (eg malaria). )), SIRS or septicemia, cancer, acute renal failure (AKI), CNS disorder (eg, seizures, neurodegenerative disease), autoimmune disease, vascular disease, hypotension, and shock.
The subject is an angiotensin receptor agonist and / or precursor thereof having an amount and / or DPP3 activity of DPP3 protein in a body fluid sample that exceeds a predetermined threshold. Angiotensin receptor agonists and / or angiotensin receptor agonists for use in the treatment of the disease in the subject according to claim 1, selected from the group comprising angiotensin I, angiotensin II, angiotensin III, and angiotensin IV, in particular angiotensin II. precursor. The amount and / or PP3 activity of the DPP3 protein is determined at least once in the body fluid sample of the subject before and / or during treatment with the angiotensin receptor agonist and / or precursor thereof. Angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in the subject according to claim 1 or 2. The determination of the amount and / or DPP3 activity of DPP3 protein determined in the body fluid sample of the subject is used as a therapeutic guide and / or therapeutic monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 1 to 3. The angiotensin receptor agonist and / or an angiotensin receptor agonist for use in the treatment of a disease in the subject according to any one of claims 1 to 4, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum. precursor. The angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of claims 1 to 5, wherein the angiotensin receptor agonist and / or a precursor thereof is administered to the subject in a pharmaceutical preparation. / Or its precursor. The angiotensin receptor agonist and / or a precursor thereof is angiotensin II acetate, and the pharmaceutical preparation is in a dry state where it is reconstituted before use, for use in the treatment of a disease in the subject according to claim 6. Angiotensin receptor agonist and / or precursor thereof. The angiotensin receptor agonist and / or its precursor is 0.1 to 200 ng / kg / min, preferably 1 to 100 ng / kg / min, more preferably 2 to 80 ng / kg / min, still more preferably 5 to Administered at a rate of 60 ng / kg / min, even more preferably 10-50 ng / kg / min, even more preferably 15-40 ng / kg / min, most preferably 20 ng / kg / min. Angiotensin II, an angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of claims 1-7. The angiotensin receptor agonist and / or a precursor thereof, particularly angiotensin II, for use in the treatment of a disease in a subject according to any one of claims 1 to 8, which is administered in combination with an inhibitor of DPP3. Angiotensin receptor agonist and / or precursor thereof. The angiotensin receptor agonist and / for use in the treatment of the disease in the subject according to claim 9, wherein the inhibitor of DPP3 is selected from the group comprising an anti-DPP3 antibody or an anti-DPP3 antibody fragment or an anti-DPP3 non-Ig scaffold. Or its precursor. Used in the treatment of a disease in a subject according to claim 9 or 10, wherein the inhibitor of DPP3 is an anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold that binds to SEQ ID NO: 1, in particular SEQ ID NO: 2. Angiotensin receptor agonist and / or its precursor for. For the treatment of a disease in a subject according to any one of claims 9 to 11, wherein the inhibitor of DPP3 is an antibody or fragment or scaffold that exhibits a minimum binding affinity of ^10-7 M or less for DPP3. Angiotensin receptor agonist and / or precursor thereof for use. Angiotensin receptor agonist and / or angiotensin receptor agonist for use in the treatment of a disease in the subject according to any one of claims 9 to 12, wherein the inhibitor of DPP3 is a unispecific antibody or fragment or scaffold. precursor. The inhibitor of DPP3 binds to full-length DPP3 and increases the activity of DPP3 by at least 10%, or at least 50%, more preferably at least 60%, even more preferably greater than 70%, even more preferably greater than 80%, further. Angiotensin receptor agonist for use in the treatment of a disease in the subject according to any one of claims 9 to 13, more preferably an antibody or fragment or scaffold that suppresses more than 90%, even more preferably more than 95%. And / or its precursor. The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, and:
The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
The step of separating DPP3 bound to the capture binder,
The claim comprises a step of quantifying the DPP3 activity by adding a substrate of DPP3 to the separated DPP3 and then measuring and quantifying the conversion of the substrate of DPP3, or a step of quantifying the amount of the DPP3 protein. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of 1 to 14. The method for determining DPP3 activity in the body fluid sample of the subject is as follows:
The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
The step of separating DPP3 bound to the capture binder,
The step of adding the substrate of DPP3 to the separated DPP3,
Angiotensin receptor agonist for use in the treatment of a disease in a subject according to any one of claims 1-15, comprising the step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3. And / or its precursor. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, antibody fragment, or. An angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of claims 1 to 16, selected from the group of non-IgG scaffolds. Claim that the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody. The angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in the subject according to any one of 1 to 17. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, and the determination comprises the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is immobilized on the surface. Angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in the subject according to any one of claims 1-18. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of interest, and the separation step is a washing step of removing components of the sample that are not bound to the capture binder from the captured DPP3. , Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of claims 16-19. DPP3 activity was determined in the body fluid sample of interest, and conversion of the DPP3 substrate was associated with fluorescence of the fluorescent substrate (eg Arg-Arg-βNA, Arg-Arg-AMC), color change of the color-developing substrate, aminoluciferin. Active staining after substrate fluorescence (Promega Protease-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis Used in the treatment of the disease in the subject according to any one of claims 16-20, detected by a method selected from the group comprising (immobilized active DPP3) or a western blot (cleavage product). Angiotensin receptor agonist and / or precursor thereof for. DPP3 activity is determined in the body fluid sample of the subject and the substrates are angiotensin II, III, IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin, proctoline, ACTH. And MSH, or the subject of any one of claims 16-21, selected from the group comprising dipeptides bound to phosphors, chromophores or enkephalin (where the dipeptide is Arg-Arg). Angiotensin receptor agonists and / or precursors thereof for use in the treatment of diseases. The DPP3 activity is determined in the body fluid sample of interest and the substrate is selected from the group comprising dipeptides bound to fluoresceins, chromophores or aminoluciferin, where the dipeptide is Arg-Arg. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of Items 16 to 22. A method or assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of interest as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. use. 24. The angiotensin receptor of claim 24, wherein the angiotensin receptor agonist and / or precursor thereof is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, and angiotensin IV, in particular angiotensin II. Use of methods or assays or kits for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of interest as a treatment guide and / or treatment monitoring for treatment with agonists and / or precursors thereof. The amount and / or the DPP3 activity of the DPP3 protein is determined at least once in the body fluid sample of the subject before and / or during treatment with the angiotensin receptor agonist and / or precursor thereof. 24 or 25, the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of interest as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. Use of methods or assays or kits to determine. Therapeutic guide for treatment with the angiotensin receptor agonist and / or precursor thereof according to any one of claims 24-26, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum. And / or use of a method or assay or kit to determine the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of interest as a therapeutic monitor. The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, and:
The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
The step of separating DPP3 bound to the capture binder,
The claim comprises a step of quantifying the DPP3 activity by adding a substrate of DPP3 to the separated DPP3 and then measuring and quantifying the conversion of the substrate of DPP3, or a step of quantifying the amount of the DPP3 protein. The amount of DPP3 protein and / or DPP3 in a body fluid sample of interest as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, as described in any one of 24-27. Use of methods or assays or kits to determine activity. The method for determining DPP3 activity in the body fluid sample of the subject is as follows:
The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
The step of separating DPP3 bound to the capture binder,
The step of adding the substrate of DPP3 to the separated DPP3,
The treatment with angiotensin receptor agonist and / or precursor thereof according to any one of claims 24-28, which comprises the step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3. Use of a method or assay or kit to determine the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of interest as a treatment guide and / or treatment monitoring for. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, antibody fragment,. Or in a body fluid sample of interest as a treatment guide and / or treatment monitoring for treatment with an angiotensin receptor agonist and / or precursor thereof, according to claim 28 or 29, selected from the group of non-IgG scaffolds. Use of methods or assays or kits to determine the amount and / or DPP3 activity of DPP3 protein. Claim that the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody. The amount of DPP3 protein and / or DPP3 in a body fluid sample of interest as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, as described in any one of 28-30. Use of methods or assays or kits to determine activity. The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, the determination comprising the use of a capture binder specifically bound to full-length DPP3, wherein the capture binder was immobilized on the surface. Of the DPP3 protein in a body fluid sample of interest as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, according to any one of claims 28-31. Use of methods or assays or kits to determine the amount and / or DPP3 activity. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, and the separation step is a washing step of removing components of the sample that are not bound to the capture binder from the captured DPP3. , The amount of DPP3 protein in a body fluid sample of interest and as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof according to any one of claims 28-32. / Or the use of a method or assay or kit to determine DPP3 activity. DPP3 activity was determined in the body fluid sample of interest, and conversion of the DPP3 substrate was associated with fluorescence of the fluorescent substrate (eg Arg-Arg-βNA, Arg-Arg-AMC), color change of the color-developing substrate, and aminoluciferin. Active staining after substrate luminescence (Promega's Protein-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis The angiotensin receptor agonist and / according to any one of claims 28-33, which is detected by a method selected from the group comprising (immobilized active DPP3) or a western blot (cleavage product). Or the use of a method or assay or kit to determine the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of interest as a treatment guide and / or treatment monitoring for treatment with its precursors. DPP3 activity is determined in the body fluid sample of the subject and the substrates are angiotensin II, III, IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin, protein, ACTH. And MSH, or any one of claims 28-34, selected from the group comprising a dipeptide bound to a fluorescent group, chromophore or enkephalin (where the dipeptide is Arg-Arg). A method or assay or kit for determining the amount and / or DPP3 activity of DPP3 protein in a body fluid sample of interest as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof. use. The DPP3 activity is determined in the body fluid sample of interest and the substrate is selected from the group comprising dipeptides bound to fluoresceins, chromophores or aminoluciferins, where the dipeptide is Arg-Arg. The amount and / or amount of DPP3 protein in a body fluid sample of interest as a treatment guide and / or treatment monitoring for treatment with angiotensin receptor agonists and / or precursors thereof, as described in any one of Items 28-35. Use of methods or assays or kits to determine DPP3 activity. Claimed that treatment with angiotensin receptor agonists and / or precursors thereof is discontinued when a patient has an amount and / or DPP3 activity of DPP3 protein in a body fluid sample below a predetermined threshold level. Angiotensin receptor agonist and / or a precursor thereof for use in the treatment of a disease in the subject according to any one of 1 to 23. The treatment with angiotensin receptor agonists and / or precursors thereof is initiated when the patient has an amount and / or DPP3 activity of DPP3 protein in the body fluid sample above a predetermined threshold level. Angiotensin receptor agonist and / or precursor thereof for use in the treatment of a disease in the subject according to any one of 23 and 37. A method for predicting the risk of adverse events in a subject, including:
The step of determining the level of DPP3 in the body fluid sample of interest;
The step of comparing the determined DPP3 level with a predetermined threshold;
The step of correlating the level of DPP3 with the risk of adverse events in the subject,
A method in which an elevated level above a certain threshold predicts an increased risk of said adverse event. The method for predicting the risk of an adverse event in a subject according to claim 39, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum. The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, and:
The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
The step of separating DPP3 bound to the capture binder,
39. Claim 39, comprising a step of quantifying the DPP3 activity by adding a substrate of DPP3 to the separated DPP3 and measuring and quantifying the conversion of the substrate of DPP3, or a step of quantifying the amount of the DPP3 protein. Or a method for predicting the risk of adverse events in the subject described in 40. The method for determining DPP3 activity in the body fluid sample of interest is:
The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
The step of separating DPP3 bound to the capture binder,
The step of adding the substrate of DPP3 to the separated DPP3,
A method for predicting the risk of adverse events in a subject according to any one of claims 39-41, comprising the step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, antibody fragment,. Alternatively, a method for predicting the risk of an adverse event in a subject according to any one of claims 39-42, selected from the group of non-IgG scaffolds. Claim that the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody. A method for predicting the risk of adverse events in the subject described in any one of 37-41. The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, the determination comprising the use of a capture binder specifically bound to full-length DPP3, wherein the capture binder was immobilized on the surface. A method for predicting the risk of an adverse event in the subject of any one of claims 39-44. In a washing step in which the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of interest and the separation step removes components in the sample that are not bound to the capture binder from the captured DPP3. A method for predicting the risk of an adverse event in the subject of any one of claims 39-45. DPP3 activity was determined in the body fluid sample of interest, and conversion of the DPP3 substrate was associated with fluorescence of the fluorescent substrate (eg Arg-Arg-βNA, Arg-Arg-AMC), color change of the color-developing substrate, aminoluciferin. Active staining after substrate fluorescence (Promega Protease-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis The risk of adverse events in the subject according to any one of claims 39-46, detected by a method selected from the group comprising (immobilized active DPP3) or western blots (cleavage products). How to predict. DPP3 activity is determined in the body fluid sample of the subject and the substrates are angiotensin II, III, IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin, proctrin, ACTH. And MSH, or the subject of any one of claims 39-47 selected from the group comprising a dipeptide bound to a fluorescent group, chromophore or enkephalin (where the dipeptide is Arg-Arg). A method for predicting the risk of adverse events in. The DPP3 activity is determined in the body fluid sample of interest and the substrate is selected from the group comprising a dipeptide bound to a fluorophore, chromophore or aminoluciferin, wherein the dipeptide is Arg-Arg. A method for predicting the risk of adverse events in the subject described in any one of paragraphs 39-48. A method for predicting the risk of organ dysfunction or deterioration of organ function in a subject.
The step of determining the level of DPP3 in the body fluid sample of interest;
The process of comparing the determined DPP3 level with the predetermined range value;
A method comprising correlating said levels of DPP3 with said risk of organ dysfunction or deterioration in the subject, wherein elevated levels above a certain range predict an increased risk of organ dysfunction or deterioration. .. The method for predicting the risk of organ dysfunction or deterioration of organ function in the subject according to claim 50, wherein the body fluid sample of the subject is selected from whole blood, plasma, and serum. The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, and:
The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
The step of separating DPP3 bound to the capture binder,
The claim comprises a step of quantifying the DPP3 activity by adding a substrate of DPP3 to the separated DPP3 and then measuring and quantifying the conversion of the substrate of DPP3, or a step of quantifying the amount of the DPP3 protein. A method for predicting the risk of organ dysfunction or deterioration of organ dysfunction in a subject according to 50 or 51. The method of determining DPP3 activity in the body fluid sample of the subject is:
The step of contacting the sample with a capture binder that specifically binds to full-length DPP3,
The step of separating DPP3 bound to the capture binder,
The step of adding the substrate of DPP3 to the separated DPP3,
Predict the risk of organ dysfunction or deterioration of organ function in the subject according to any one of claims 50-52, comprising the step of quantifying the DPP3 activity by measuring and quantifying the conversion of the substrate of DPP3. How to. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody, antibody fragment,. Alternatively, a method for predicting the risk of organ dysfunction or deterioration of organ function in the subject according to any one of claims 50-53, selected from the group of non-IgG scaffolds. Claim that the amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of the subject, the determination comprising the use of a capture binder that specifically binds to full-length DPP3, wherein the capture binder is an antibody. A method for predicting the risk of organ dysfunction or deterioration of organ function in the subject according to any one of 50 to 54. The amount and / or DPP3 activity of the DPP3 protein was determined in the body fluid sample of the subject, the determination comprising the use of a capture binder specifically bound to full-length DPP3, wherein the capture binder was immobilized on the surface. A method for predicting the risk of organ dysfunction or deterioration of organ function in the subject according to claims 50 to 55. The amount and / or DPP3 activity of the DPP3 protein is determined in the body fluid sample of interest, and the separation step is a washing step of removing components of the sample that are not bound to the capture binder from the captured DPP3. , A method for predicting the risk of organ dysfunction or deterioration of organ function in the subject according to any one of claims 50 to 56. DPP3 activity was determined in the body fluid sample of the subject, and conversion of the DPP3 substrate was associated with fluorescence of the fluorescent substrate (eg Arg-Arg-βNA, Arg-Arg-AMC), color change of the color-developing substrate, aminoluciferin. Active staining after substrate fluorescence (Promega Protease-Glo ™ assay), mass analysis, HPLC / FPLC (reverse phase chromatography, size exclusion chromatography), thin layer chromatography, capillary zone electrophoresis, gel electrophoresis (Immobilized active DPP3), or the risk of organ dysfunction or deterioration of organ function in the subject according to claims 50-57, detected by a method selected from the group comprising Western blots (cleavage products). How to predict. DPP3 activity is determined in the body fluid sample of the subject and the substrates are angiotensin II, III, IV, Leu-enkephalin, Met-enkephalin, endomorphins 1 and 2, barolphin, β-casomorphin, dynorphin, proctrin, ACTH. And MSH, or the subject of any one of claims 50-58 selected from the group comprising a dipeptide bound to a fluorescent group, chromophore or enkephalin (where the dipeptide is Arg-Arg). A method for predicting the risk of organ dysfunction or deterioration of organ function in. The DPP3 activity is determined in the body fluid sample of interest and the substrate is selected from the group comprising a dipeptide bound to a fluorophore, chromophore or aminoluciferin, wherein the dipeptide is Arg-Arg. A method for predicting the risk of organ dysfunction or deterioration of organ function in the subject according to any one of Items 50 to 59.

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